Short interfering ribonucleic acid (siRNA) for oral administration

ABSTRACT

Short interfering ribonucleic acid (siRNA) for oral administration, said siRNA comprising two separate RNA strands that are complementary to each other over at least 15 nucleotides, wherein each strand is 49 nucleotides or less, and wherein at least one of which strands contains at least one chemical modification.

PRIORITY INFORMATION

This U.S. application is a divisional application of Ser. No.13/651,808, filed Oct. 15, 2012, which is a divisional applicationclaiming priority to U.S. Pat. No. 8,344,128, issued Jan. 1, 2013, whichis a divisional application claiming priority U.S. PCT application Ser.No. 12/299,396, filed 2 May 2007, which claims priority to PCTApplication Serial No. PCT/EP07/003867, filed 2 May 2007, which claimspriority to GB Application Serial No. 0608838.9, filed 4 May 2006, thecontents of which are incorporated herein by reference in theirentirety.

BACKGROUND

RNA interference initially discovered in plants as Post-TranscriptionalGene Silencing (PTGS), is a highly conserved mechanism triggered bydouble-stranded RNA (dsRNA) and able to down regulate transcript ofgenes homologous to the dsRNA¹. The dsRNA is first processed by Dicerinto short duplexes of 21-23 nt, called short interfering RNAs(siRNAs)². Incorporated in RNA-induced silencing complex (RISC) they areable to mediate gene silencing through cleavage of the target mRNA inthe center of the region of homology by Argonaute 2, a component ofRISC³. In 2001, Elbashir et al⁴ demonstrated that the directintroduction of synthetic siRNAs would mediate RNA interference genesilencing in drosophila but also in mammalian cells. Since then,siRNA-mediated gene silencing has become a powerful and widely-usedmolecular biology tool in both target identification target validationstudies. Use of siRNAs for gene silencing in animal studies has beendescribed in a limited amount of animal models. Unmodified siRNAs weredelivered locally in the eye⁵, intrathecally or intracerebellarly in thecentral nervous system⁶, and intranasally for the inhibition ofrespiratory viruses⁷. Intravenous hydrodynamic tail vein injection ofunmodified siRNAs has also been studied. This approach allows a rapiddelivery, mainly to the liver⁸. A very limited number of studies havebeen reported on the systemic administration of unmodified siRNAs.Duxbury et al⁹ administered intravenously unmodified siRNAs targetingFocal Adhesion Kinase to an orthotopic tumor xenograft mice model, andobserved a tumor growth inhibition as well as a chemosensitization togemcitabine. Soutscheck et al reported the systemic use of highlychemically modified siRNAs for the endogeneous silencing ApolipoproteinB. Intraperitoneal administration of most anti-ApoB siRNA at the highdose of 50 mg/kg reduced ApoB protein level and Lipoproteinconcentration¹⁰. Despite these examples, in vivo use of siRNAs uponsystemic delivery requires improvements in order to make this technologywidely applicable for target validation or therapeutic applications.Indeed, unmodified siRNAs are subject to enzymatic digestion, mainly bynucleases abundant in the blood stream. In order to improvepharmacological properties of siRNAs several groups investigatedchemical modification of these reagents. While the approaches describedare very different among themselves and that no systematic study was yetperformed, an overview of the results allows to determine the toleranceof siRNAs to chemical modifications. Several chemistries such asphosphorothioates¹¹ or boranophosphates¹², 2′-O-Methyl¹³, 2′-O-allyl¹⁴,2′-methoxyethyl (MOE) and 2′-deoxyfluoronucleotides¹⁵ or Locked NucleicAcids (LNA)¹⁶ have been investigated. These studies highlighted thattolerance for modification is not only chemistry-dependent, but alsoposition-dependent.

The present invention provides a minimally modified siRNA with improvedpharmacological properties. The minimally modified siRNAs are 19 bpdouble-stranded RNA modified on the 3′-end of each strand in order toprevent 3′-exonuclease digestion: the 3′-dideoxynucleotide overhang of21-nt siRNA has been replaced by a universal 3′-hydroxypropylphosphodiester moiety and the modification of the two first base-pairingnucleotides on 3′-end of each strand further enhances serum stability.Applied intraperitoneally or orally to adult mice, the modified siRNAsdisplayed higher potency in a growth factor induce angiogenesis modelwhich correlates with their increased serum stability.

SUMMARY

In one aspect, the present invention provides a short interferingribonucleic acid (siRNA) for oral administration, said siRNA comprisingtwo separate RNA strands that are complementary to each other over atleast 15 nucleotides, wherein each strand is 49 nucleotides or less, andwherein at least one of which strands contains at least one chemicalmodification.

In one embodiment, the siRNA comprises at least one modified nucleotide.

In another embodiment, the siRNA comprises at least one 3′ end cap.

In another embodiment, said modified nucleotide is selected from among2′ alkoxyribonucleotide, 2′ alkoxyalkoxy ribonucleotide, a lockednucleic acid ribonucleotide (LNA), 2′-fluoro ribonucleotide, morpholinonucleotide.

In another embodiment, said modified nucleotide is selected from amongnucleotides having a modified internucleoside linkage selected fromamong phosphorothioate, phosphorodithioate, phosphoramidate,boranophosphonoate, and amide linkages.

In another embodiment, said two RNA strands are fully complementary toeach other.

In another embodiment, said siRNA comprises a 1 to 6 nucleotide overhangon at least one of the 5′ end or 3′ end.

In another embodiment, the siRNA contains at least one 3′ cap, which ischemical moiety conjugated to the 3′ end via the 3′ carbon and isselected from among compounds of Formula I:

-   -   wherein    -   X is O or S    -   R₁ and R₂ are independently OH, NH₂, SH, alkyl, aryl,        alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl,        aryl-alkyl can be substituted by additional heteroatoms and        functional groups, preferably a heteroatom selected from the        group of N, O, or S or a functional group selected from the        group OH, NH₂, SH, carboxylic acid or ester;    -   or R₁ and R₂ may be of formula Y—Z where Y is O, N, S and Z is        H, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl,        alkyl-aryl, aryl-alkyl can be substituted by additional        heteroatoms, preferably a heteroatom selected from the group of        N, O, or S.

In another embodiment, the siRNA contains at least one strand which iscomplementary over at least 15 nucleotides to the mRNA or pre-mRNA ofVEGFR-1, VEGFR-2, VEGFR3, Tie2, bFGFR, IL8RA, IL8RB, Fas, or IGF2R.

In another embodiment, the siRNA contains at least one strand whichcomprises a sequence selected from SEQ ID NO 1-900.

In another embodiment, the siRNA is chosen from the group consisting ofSEQ ID NO 901-930.

In another embodiment, the siRNA has a stability in a standard gastricacid assay that is greater than an unmodified siRNA with the samenucleotide sequence.

In another embodiment, the siRNA has a stability in a standard gastricacid assay that is greater than or equal to 50% after 30 minutesexposure.

In another embodiment, the siRNA has a stability in a standard serumassay greater than unmodified siRNA.

In another embodiment, the siRNA has a stability in a standard serumassay that is greater than or equal to 50% after 30 minutes exposure.

In another embodiment, the siRNA has a stability in a standardintestinal lavage assay that is greater than unmodified siRNA.

In another embodiment, the siRNA has an enhanced oral bioavailabilitycompared to an unmodified siRNA of the same nucleotide sequence.

In one aspect, the invention provides a pharmaceutical compositioncomprising an siRNA with any one or more of the above properties.

In another aspect, the invention provides an siRNA with any one or moreof the above properties for use as a medicament.

In another aspect, the invention provides the use of an siRNA with anyone or more of the above properties in the preparation of a medicamentfor treating an angiogenic disorder.

In another aspect, the invention provides the use of an siRNA with anyone or more of the above properties to inhibit an angiogenic process invitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1b, 1c, 1d and 1e : Metabolic degradation of unmodified siRNApGl3-siRNA (wild-type siRNA in mouse serum); a-c) Ion Exchange-HPLCanalysis of unmodified siRNAs after incubation in mouse serum for 0′,30′ and 180′; After 30′ of incubation at 37° C., major peak in the IonExchange HPLC was isolated and re-injected in LC-MS, d) table ofdetected molecular weights and their assignments; e) ESI-MS spectrum

FIG. 2: illustration of four double-stranded RNA formats: wild-type (orunmodified) siRNA. MOE o/h siRNA, C3-siRNA and C3-MOE siRNA.

FIG. 3: Stability of siRNA in 3 different formats in mouse gastric acid.Samples were incubated at 37° C. in mouse gastric acid at a 2 micromolarconcentration. Disappearance of parent compound was followed over a 2-6hours period by quantifying the parent compound band.

-   Lane 1-7: wild-type siRNA in gastric acid at t=0, 5, 10, 15, 30, 60    and 120 min-   Lane 8: ds RNA ladder (30, 21, 19, 16, 13, 10 bp)-   Lane 9-15: C3 siRNA in gastric acid at t=0, 5, 10, 15, 30, 60 and    120 min-   Lane 16: ds RNA ladder (30, 21, 19, 16, 13, 10 bp)-   Lane 17-24: C3-MOE siRNA in gastric acid at t=0, 5, 10, 15, 30, 60    and 120 min

FIG. 4: Stability of siRNA in 4 different formats in intestinal lavage.Samples were incubated at 37° C. in liver microsomes at a 5 micromolarconcentration.

(From left to right)

-   Lane 1: ds RNA ladder (30, 21, 19, 16, 13, 10 bp)-   Lane 2-7: wild-type siRNA in intestinal lavage at t=0, 15, 30, 60,    180 and 360 min-   Lane 8-13: moe o/h siRNA in intestinal lavage at t=0, 15, 30, 60,    180 and 360 min-   Lane 14-19: C3 siRNA in intestinal lavage at t=0, 15, 30, 60, 180    and 360 min-   Lane 20-25: C3-MOE siRNA in intestinal lavage at t=0, 15, 30, 60,    180 and 360 min

FIG. 5: Stability of siRNA in 4 different formats in liver microsomes.Samples were incubated at 37° C. in intestinal fluid from rat intestinallavage at a 2 micromolar concentration.

(From left to right)

-   Lane 1: ds

FIG. 6: Stability of siRNA in 4 different formats in mouse serum.Samples were incubated at 37° C. in mouse serum at a 2 micromolarconcentration. Disappearance of parent compound was followed over a 6hours period by quantifying the parent compound band.

(From left to right)

-   Lane 1: ds RNA ladder (30, 21, 19, 16, 13, 10 bp) RNA ladder (30,    21, 19, 16, 13, 10 bp)-   Lane 2: wild-type siRNA untreated-   Lane 3: moe o/h siRNA untreated-   Lane 4: C3 siRNA untreated-   Lane 5: C3-MOE siRNA untreated-   Lane 6-9: same as 2-5 in liver microsomes t=0-   Lane 10-13: same as 2-5 in liver microsomes t=60′-   Lane 14-17: same as 2-5 in supernatant S12 t=0-   Lane 18-21: same as 2-5 in supernatant S12 t=60′-   Lane 2-7: wild-type siRNA in mouse serum at t=0, 15, 30, 60, 180 and    360 min-   Lane 8-13: moe o/h siRNA in mouse serum at t=0, 15, 30, 60, 180 and    360 min-   Lane 14-19: C3 siRNA in mouse serum at t=0, 15, 30, 60, 180 and 360    min-   Lane 20-25: C3-MOE siRNA mouse serum at t=0, 15, 30, 60, 180 and 360    min

FIG. 7: Characterization in cellulo of 3 formats of anti-VEGFR2 siRNA (2independent sequences). Wild-type siRNA, C3-siRNA and C3-MOE siRNA weretransfected into MS1 cells at three concentrations (1, 5, 10 nM).Silencing potency was assessed by measuring VEGFR2 cell surface level byFACS.

FIGS. 8a and 8b : In vivo testing of wild-type siRNA, C3-siRNA andC3-Moe siRNA in a growth factor induced angiogenesis “Agar Chamber”mouse model. FIG. 8a shows the results of controls, unmodified VEGFR2siRNA and C3 modified VEGFR2 siRNA at 1, 5 and 25 micrograms per mouseper day. FIG. 8b shows controls, C3 modified VEGFR2 siRNA and of C3-MOEVEGFR2 siRNA at 0.2, 1 and 5 micrograms per mouse per day. In each casepools of 2 anti-VEGFR2 siRNAs were given daily intraperitoneally forthree days.

FIGS. 9a and 9b : In vivo testing of anti-VEGFR2 C3-MOE siRNA givenintraperitoneally (i.p.) in a B16 homograft melanoma tumor mouse modelat 5 and 20 micrograms per mouse per day. FIG. 9a shows that i.p.treatment with modified VEGFR2 siRNA significantly reduces tumourdevelopment. FIG. 9b also shows that i.p. injection of VEGFR2 siRNA at20 ug per mouse results in significant inhibition of tumour growth.

FIG. 10: In vivo testing of C3-MOE siRNA in a growth factor inducedangiogenesis mouse model. anti-VEGFR2 siRNAs were given daily orally forthree days at 20 micrograms per mouse per day.

FIGS. 11a and 11b : In vivo testing of C3-MOE siRNA in a growth factorinduced angiogenesis mouse model. anti-Tie2 siRNAs were given dailyintraperitoneally (1 and 0.2 micrograms per mouse per day) or orally (20and 5 micrograms per mouse per day) for three days. FIG. 11a : weight ofexcised tissue; FIG. 11b : Tie2 protein knock-down

DETAILED DISCLOSURE OF THE INVENTION

The present invention relates to compositions and methods for treatingangiogenic disorders in a mammal. Specifically, the invention relates tosmall-interfering RNA (“siRNA”) which may be used to treat angiogenicdisorders upon oral administration to a mammal.

Angiogenesis targets in vascular endothelial cells include the followingtargets/genes: VEGFR-1 (GenBank Accession # AF06365); VEGFR-2 (GenBankAccession # AF063658); VEGFR-3 (GenBank Accession # (NM_002020); Tie2(TEK) (GenBank Accession # NM_000459); bFGFR (GenBank Accession #M60485); IL8RA (GenBank Accession # L19591); IL8RB (GenBank Accession #L19593); Fas (GenBank Accession # X89101); IGF2R (GenBank Accession #NM_000876).

The siRNA molecules according to the present invention mediate RNAinterference (“RNAi”). The term “RNAi” is well known in the art and iscommonly understood to mean the inhibition of one or more target genesin a cell by siRNA with a region which is complementary to the targetgene. Various assays are known in the art to test siRNA for its abilityto mediate RNAi (see for instance Elbashir et al., Methods 26 (2002),199-213). The effect of the siRNA according to the present invention ongene expression will typically result in expression of the target genebeing inhibited by at least 10%, 33%, 50%, 90%, 95% or 99% when comparedto a cell not treated with the RNA molecules according to the presentinvention.

“siRNA” or “small-interfering ribonucleic acid” according to theinvention has the meanings known in the art, including the followingaspects. The siRNA consists of two strands of ribonucleotides whichhybridize along a complementary region under physiological conditions.The strands are separate but they may be joined by a molecular linker incertain embodiments. The individual ribonucleotides may be unmodifiednaturally occurring ribonucleotides, unmodified naturally occurringdeoxyribonucleotides or they may be chemically modified or synthetic asdescribed elsewhere herein.

The siRNA molecules in accordance with the present invention comprise adouble-stranded region which is substantially identical to a region ofthe mRNA of the target gene. A region with 100% identity to thecorresponding sequence of the target gene is suitable. This state isreferred to as “fully complementary”. However, the region may alsocontain one, two or three mismatches as compared to the correspondingregion of the target gene, depending on the length of the region of themRNA that is targeted, and as such may be not fully complementary. In anembodiment, the RNA molecules of the present invention specificallytarget one given gene. In order to only target the desired mRNA, thesiRNA reagent may have 100% homology to the target mRNA and at least 2mismatched nucleotides to all other genes present in the cell ororganism. Methods to analyze and identify siRNAs with sufficientsequence identity in order to effectively inhibit expression of aspecific target sequence are known in the art. Sequence identity may beoptimized by sequence comparison and alignment algorithms known in theart (see Gribskov and Devereux, Sequence Analysis Primer, StocktonPress, 1991, and references cited therein) and calculating the percentdifference between the nucleotide sequences by, for example, theSmith-Waterman algorithm as implemented in the BESTFIT software programusing default parameters (e.g., University of Wisconsin GeneticComputing Group).

Another factor affecting the efficiency of the RNAi reagent is thetarget region of the target gene. The region of a target gene effectivefor inhibition by the RNAi reagent may be determined by experimentation.A suitable mRNA target region would be the coding region. Also suitableare untranslated regions, such as the 5′-UTR, the 3′-UTR, and splicejunctions. For instance, transfection assays as described in Elbashir S.M. et al, 2001 EMBO J., 20, 6877-6888 may be performed for this purpose.A number of other suitable assays and methods exist in the art which arewell known to the skilled person.

The length of the region of the siRNA complementary to the target, inaccordance with the present invention, may be from 10 to 100nucleotides, 12 to 25 nucleotides, 14 to 22 nucleotides or 15, 16, 17 or18 nucleotides. Where there are mismatches to the corresponding targetregion, the length of the complementary region is generally required tobe somewhat longer.

Because the siRNA may carry overhanging ends (which may or may not becomplementary to the target), or additional nucleotides complementary toitself but not the target gene, the total length of each separate strandof siRNA may be 10 to 100 nucleotides, 15 to 49 nucleotides, 17 to 30nucleotides or 19 to 25 nucleotides.

The phrase “each strand is 49 nucleotides or less” means the totalnumber of consecutive nucleotides in the strand, including all modifiedor unmodified nucleotides, but not including any chemical moieties whichmay be added to the 3′ or 5′ end of the strand. Short chemical moietiesinserted into the strand are not counted, but a chemical linker designedto join two separate strands is not considered to create consecutivenucleotides.

The phrase “a 1 to 6 nucleotide overhang on at least one of the 5′ endor 3′ end” refers to the architecture of the complementary siRNA thatforms from two separate strands under physiological conditions. If theterminal nucleotides are part of the double-stranded region of thesiRNA, the siRNA is considered blunt ended. If one or more nucleotidesare unpaired on an end, an overhang is created. The overhang length ismeasured by the number of overhanging nucleotides. The overhangingnucleotides can be either on the 5′ end or 3′ end of either strand.

The siRNA according to the present invention confer a high in vivostability suitable for oral delivery by including at least one modifiednucleotide in at least one of the strands. Thus the siRNA according tothe present invention contains at least one modified or non-naturalribonucleotide. A lengthy description of many known chemicalmodifications are set out in published PCT patent application WO200370918 and will not be repeated here. Suitable modifications for oraldelivery are more specifically set out in the Examples and descriptionherein. Suitable modifications include, but are not limited tomodifications to the sugar moiety (i.e. the 2′ position of the sugarmoiety, such as for instance 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin etal., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group)or the base moiety (i.e. a non-natural or modified base which maintainsability to pair with another specific base in an alternate nucleotidechain). Other modifications include so-called ‘backbone’ modificationsincluding, but not limited to, replacing the phosphoester group(connecting adjacent ribonuclotides with for instance phosphorothioates,chiral phosphorothioates or phosphorodithioates). Finally, endmodifications sometimes referred to herein as 3′ caps or 5′ caps may beof significance. As illustrated in Table 1, caps may consist of simplyadding additional nucleotides, such as “T-T” which has been found toconfer stability on an siRNA. Caps may consist of more complexchemistries which are known to those skilled in the art.

In an embodiment used in the Examples below, the 3′ cap is a chemicalmoiety conjugated to the 3′ end via the 3′ carbon and is selected fromamong compounds of Formula I:

wherein

-   X is O or S-   R₁ and R₂ are independently OH, NH₂, SH, alkyl, aryl, alkyl-aryl,    aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be    substituted by additional heteroatoms and functional groups,    preferably a heteroatom selected from the group of N, O, or S or a    functional group selected from the group OH, NH₂, SH, carboxylic    acid or ester;-   or R₁ and R₂ may be of formula Y—Z where Y is O, N, S and Z is H,    alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl,    aryl-alkyl can be substituted by additional heteroatoms, preferably    a heteroatom selected from the group of N, O, or S.

Examples of modifications on the sugar moiety include 2′alkoxyribonucleotide, 2′ alkoxyalkoxy ribonucleotide, locked nucleicacid ribonucleotide (LNA), 2′-fluoro ribonucleotide, morpholinonucleotide.

The internucleoside linkage may also be modified. Examples ofinternucleoside linkage include phosphorothioate, phosphorodithioate,phosphoramidate, and amide linkages.

R₁ may be OH.

R₁ and R₂ together may comprise from 1 to 24 C-atoms, from 1 to 12C-atoms, from 2 to 10 C-atoms, from 1 to 8 or from 2 to 6 C-atoms. Inanother embodiment, R₁ and R₂ are independently OH, lower alkyl, loweraryl, lower alkyl-aryl, lower aryl-alkyl, where lower alkyl, lower aryl,lower alkyl-aryl, lower aryl-alkyl can be substituted by additionalheteroatoms and functional groups as defined above. In anotherembodiment, R₁ and R₂ are not both OH.

The term “lower” in connection with organic radicals or compounds meansa compound or radical which may be branched or unbranched with up to andincluding 7 carbon atoms, preferably 1-4 carbon atoms. Lower alkylrepresents, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, tert-butyl, n-pentyl and branched pentyl, n-hexyl andbranched hexyl.

Examples of alkoxys include O-Met, O-Eth, O-prop, O-but, O-pent, O-hex.

Methods for the synthesis of siRNA, including siRNA containing at leastone modified or non-natural ribonucleotides are well known and readilyavailable to those of skill in the art. For example, a variety ofsynthetic chemistries are set out in published PCT patent applicationsWO2005021749 and WO200370918, both incorporated herein by reference. Thereaction may be carried out in solution or, preferably, on solid phaseor by using polymer supported reagents, followed by combining thesynthesized RNA strands under conditions, wherein a siRNA molecule isformed, which is capable of mediating RNAi.

The present invention provides an siRNA containing at least one modifiednucleotide which is suitable for oral delivery. In functional terms thismeans siRNA will have suitable pharmacokinetics and biodistribution uponoral administration to achieve delivery to the target tissue of concern.In particular this requires serum stability, lack of immune response,and drug like behaviour. Many of these features of siRNA can beanticipated based on the standard gastric acid assays and standard serumassays disclosed elsewhere herein.

In another aspect, the present invention provides methods for theinhibition of a target gene comprising introducing into a cell and siRNAaccording to the present invention, which is capable of inhibiting atleast one target gene by RNAi. Also, more than one species of siRNA,which are each specific for another target region, may be introducedinto a cell at the same time or sequentially.

The present invention is not limited to any type of target gene ornucleotide sequence. For example, the target gene can be a cellulargene, an endogenous gene, a pathogen-associated gene, a viral gene or anoncogene. Angiogenic genes are of particular importance to the inventionbecause some of the Examples highlight that the orally delivered siRNAof the invention may accumulate at sites of vasculogenesis,neovascularization or angiogenesis. An updated listing of angiogenicgenes at these sites of particular interest for the invention are listedin AngioDB: database of angiogenesis and angiogenesis-related moleculesTae-Kwon Sohn, Eun-Joung Moonl, Seok-Ki Leel, Hwan-Gue Cho2 and Kyu-WonKim3, Nucleic Acids Research, 2002, Vol. 30, No. 1 369-371 and online athttp://angiodb.snu.ac.kr/. Genes of particular significance have beenanalyzed in detail and are set out elsewhere herein.

In another aspect, the invention also provides a kit comprising reagentsfor inhibiting expression of a target gene in a cell, wherein said kitcomprises dsRNA according to the present invention. The kit comprises atleast one of the reagents necessary to carry out the in vitro or in vivointroduction of the dsRNA according to the present invention to testsamples or subjects. In a preferred embodiment, such kits also compriseinstructions detailing the procedures by which the kit components are tobe used.

“Treatment of an angiogenic disorder” as used in this disclosure meansuse of a modified siRNA of the invention in a pharmaceutical compositionfor the treatment of diseases involving the physiological andpathological processes of neovascularization, vasculogenesis and/orangiogenesis. As such, these pharmaceutical compositions are useful fortreating diseases, conditions and disorders that require inhibition ofneovascularization, vasculogenesis or angiogenesis, including but notlimited to cancer tumour growth and metastasis, neoplasm, ocularneovascularization (including macular degeneration, diabeticretinopathy, ischemic retinopathy, retinopathy of prematurity, choroidalneovascularization), rheumatoid arthritis, osteoarthritis, chronicasthma, spectic shock, inflammatory diseases, synovitis, bone andcartilage destruction, pannus growth, osteophyte formation,osteomyelitis, psoriasis, obesity, haemangioma, Kaposi's sarcoma,atherosclerosis (including atherosclerotic plaque rupture),endometriosis, warts, excess hair growth, scar keloids, allergic oedema,dysfunctional uterine bleeding, follicular cysts, ovarianhyperstimulation, endometriosis, osteomyelitis, inflammatory andinfectious processes (hepatitis, pneumonia, glumerulonephtritis),asthma, nasal polyps, transplantation, liver regeneration, leukomalacia,thyroiditis, thyroid enlargement, lymphoproliferative disorders,haematologic malignancies, vascular malformations, and pre-eclampsia.

As used herein, “treatment” means an action taken to inhibit or reduce aprocess of a disease, disorder or condition, to inhibit or reduce asymptom of a disease, disorder or condition, or to prophylacticallyprevent the onset or further development of a disease, disorder orcondition. “Treat” is the cognitive verb thereof.

An effective dose of the therapeutic agent of the invention is that doserequired to treat a disease state. The effective dose depends on thetype of disease, the composition used, the route of administration, thetype of mammal being treated, the physical characteristics of thespecific mammal under consideration, concurrent medication, and otherfactors that those skilled in the medical arts will recognize.Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day ofsiRNA is administered dependent upon potency. The nucleic acid moleculesof the invention and formulations thereof can be administered orally,topically, parenterally, by inhalation or spray, or rectally in dosageunit formulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and/or vehicles. The term parenteral asused herein includes percutaneous, subcutaneous, intravascular (e.g.,intravenous), intramuscular, intraperitoneal, or intrathecal injection,or infusion techniques and the like. In addition, there is provided apharmaceutical formulation comprising a nucleic acid molecule of theinvention and a pharmaceutically acceptable carrier. One or more nucleicacid molecules of the invention can be present in association with oneor more non-toxic pharmaceutically acceptable carriers and/or diluentsand/or adjuvants, and if desired other active ingredients. Thepharmaceutical compositions containing nucleic acid molecules of theinvention can be in a form suitable for oral use, for example, astablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsion, hard or soft capsules, or syrups orelixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. Formulations for oral use can also bepresented as hard gelatin capsules wherein the active ingredient ismixed with an inert solid diluent, for example, calcium carbonate,calcium phosphate or kaolin, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin or olive oil. Aqueous suspensions containthe active materials in a mixture with excipients suitable for themanufacture of aqueous suspensions.

Oral administration of the compositions of the invention include allstandard techniques for administering substances directly to the stomachor gut, most importantly by patient controlled swallowing of the dosageform, but also by other mechanical and assisted means of such delivery.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient. It is understood that the specific dose level for anyparticular subject depends upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

Therapeutic effect of the therapeutic agents of the invention may beenhanced by combination with other agents. Typically such other agentswill include agents known for use in treating similar diseases, such asangiogenic disorders. Alternatively, such agents may be used to reduceside-effects or unwanted effects caused by the therapeutic agents of theinvention.

The siRNA of the invention also have important research uses. One suchstudy includes research into an angiogenic process in vitro. By“angiogenic process in vitro” is meant any process for studyingangiogenesis or vasculogenesis which does not employ a whole animal. Assuch, in vitro or ex vivo methods and assays which study the steps ofthe angiogenic process using markers or indicators of angiogenesis areincluded hereby.

RNA Strand Nucleotide Sequences

The siRNA strand sequences identified in Table 1 have been identified assuitable siRNA sequences against the following targets: VEGFR-1 (GenBankAccession # AF06365); VEGFR-2 (GenBank Accession # AF063658); VEGFR-3(GenBank Accession # (NM_002020); Tie2 (TEK) (GenBank Accession #NM_000459); bFGFR (GenBank Accession # M60485); IL8RA (GenBank Accession# L19591); IL8RB (GenBank Accession # L19593); Fas (GenBank Accession #X89101); IGF2R (GenBank Accession # NM 000876).

TABLE 1  siRNAs against human VEGFR-1, VEGFR-2, VEGFR-3, Tie2, bFGFR,IL8RA, IL8RB, Fas, IGF2R SEQ SEQ Target ID ID Name possiRNA guide sequence NO siRNA complement NO VEGFR-1 1731UAUAAGAACUUGUUAACUGTG 1 CAGUUAACAAGUUCUUAUATT 451 VEGFR-1 1021UACGGUUUCAAGCACCUGCTG 2 GCAGGUGCUUGAAACCGUATT 452 VEGFR-1 1209UUUAUGCUCAGCAAGAUUGTA 3 CAAUCUUGCUGAGCAUAAATT 453 VEGFR-1 2904UUAUCUUCCUGAAAGCCGGAG 4 CCGGCUUUCAGGAAGAUAATT 454 VEGFR-1 1363UUGAGGGAUACCAUAUGCGGT 5 CGCAUAUGGUAUCCCUCAATT 455 VEGFR-1 1158UUGAUAAUUAACGAGUAGCCA 6 GCUACUCGUUAAUUAUCAATT 456 VEGFR-1 1091UUAACCAUACAACUUCCGGCG 7 CCGGAAGUUGUAUGGUUAATT 457 VEGFR-1 471UUAGGUGACGUAACCCGGCAG 8 GCCGGGUUACGUCACCUAATT 458 VEGFR-1 2751UUGCUCUUGAGGUAGUUGGAG 9 CCAACUACCUCAAGAGCAATT 459 VEGFR-1 636UUUGUCUUAUACAAAUGCCCA 10 GGCAUUUGUAUAAGACAAATT 460 VEGFR-1 1254UUGACAAUUAGAGUGGCAGTG 11 CUGCCACUCUAAUUGUCAATT 461 VEGFR-1 2375UUAUAAUUGAUAGGUAGUCAG 12 GACUACCUAUCAAUUAUAATT 462 VEGFR-1 3536UUGAGUAUGUAAACCCACUAT 13 AGUGGGUUUACAUACUCAATT 463 VEGFR-1 2971UUCCAUAGUGAUGGGCUCCTT 14 GGAGCCCAUCACUAUGGAATT 464 VEGFR-1 1774UCUGUUAUUAACUGUCCGCAG 15 GCGGACAGUUAAUAACAGATT 465 VEGFR-1 3494UUGGGAUGUAGUCUUUACCAT 16 GGUAAAGACUACAUCCCAATT 466 VEGFR-1 2269UGUUAGAGUGAUCAGCUCCAG 17 GGAGCUGAUCACUCUAACATT 467 VEGFR-1 525UUUCCAUCAGGGAUCAAAGTG 18 CUUUGAUCCCUGAUGGAAATT 468 VEGFR-1 769UUGAACUCUCGUGUUCAAGGG 19 CUUGAACACGAGAGUUCAATT 469 VEGFR-1 2246UAGACUUGUCCGAGGUUCCTT 20 GGAACCUCGGACAAGUCUATT 470 VEGFR-1 732UUGAGGACAAGAGUAUGGCCT 21 GCCAUACUCUUGUCCUCAATT 471 VEGFR-1 3813UUACUGGUUACUCUCAAGUCA 22 ACUUGAGAGUAACCAGUAATT 472 VEGFR-1 3925UUCCAGCUCAGCGUGGUCGTA 23 CGACCACGCUGAGCUGGAATT 473 VEGFR-1 1414UGCUUCGGAAUGAUUAUGGTT 24 CCAUAAUCAUUCCGAAGCATT 474 VEGFR-1 615UUGACUGUUGCUUCACAGGTC 25 CCUGUGAAGCAACAGUCAATT 475 VEGFR-1 3300UCAUCCAUUUGUACUCCUGGG 26 CAGGAGUACAAAUGGAUGATT 476 VEGFR-1 2845UGGUUUCUUGCCUUGUUCCAG 27 GGAACAAGGCAAGAAACCATT 477 VEGFR-1 2802UUAGGCUCCAUGUGUAGUGCT 28 CACUACACAUGGAGCCUAATT 478 VEGFR-1 1564UCUAGAGUCAGCCACAACCAA 29 GGUUGUGGCUGACUCUAGATT 479 VEGFR-1 1154UAAUUAACGAGUAGCCACGAG 30 CGUGGCUACUCGUUAAUUATT 480 VEGFR-1 1090UAACCAUACAACUUCCGGCGA 31 GCCGGAAGUUGUAUGGUUATT 481 VEGFR-1 1260UUCACAUUGACAAUUAGAGTG 32 CUCUAAUUGUCAAUGUGAATT 482 VEGFR-1 3530AUGUAAACCCACUAUUUCCTG 33 GGAAAUAGUGGGUUUACAUTT 483 VEGFR-1 1177AUCCUCUUCAGUUACGUCCTT 34 GGACGUAACUGAAGAGGAUTT 484 VEGFR-1 1193UUGUAUAAUUCCCUGCAUCCT 35 GAUGCAGGGAAUUAUACAATT 485 VEGFR-1 1092UUUAACCAUACAACUUCCGGC 36 CGGAAGUUGUAUGGUUAAATT 486 VEGFR-1 627UACAAAUGCCCAUUGACUGTT 37 CAGUCAAUGGGCAUUUGUATT 487 VEGFR-1 474AUGUUAGGUGACGUAACCCGG 38 GGGUUACGUCACCUAACAUTT 488 VEGFR-1 2761UAAGUCACGUUUGCUCUUGAG 39 CAAGAGCAAACGUGACUUATT 489 VEGFR-1 2752UUUGCUCUUGAGGUAGUUGGA 40 CAACUACCUCAAGAGCAAATT 490 VEGFR-1 3516UUUCCUGUCAGUAUGGCAUTG 41 AUGCCAUACUGACAGGAAATT 491 VEGFR-1 1790UACUGUAGUGCAUUGUUCUGT 42 AGAACAAUGCACUACAGUATT 492 VEGFR-1 1155AUAAUUAACGAGUAGCCACGA 43 GUGGCUACUCGUUAAUUAUTT 493 VEGFR-1 1370UUGUAGGUUGAGGGAUACCAT 44 GGUAUCCCUCAACCUACAATT 494 VEGFR-1 2227UUGAACAGUGAGGUAUGCUGA 45 AGCAUACCUCACUGUUCAATT 495 VEGFR-1 3481UUUACCAUCCUGUUGUACATT 46 UGUACAACAGGAUGGUAAATT 496 VEGFR-1 1261UUUCACAUUGACAAUUAGAGT 47 UCUAAUUGUCAAUGUGAAATT 497 VEGFR-1 1791AUACUGUAGUGCAUUGUUCTG 48 GAACAAUGCACUACAGUAUTT 498 VEGFR-1 3805UACUCUCAAGUCAAUCUUGAG 49 CAAGAUUGACUUGAGAGUATT 499 VEGFR-1 2764AAAUAAGUCACGUUUGCUCTT 50 GAGCAAACGUGACUUAUUUTT 500 VEGFR-2 617UAAUAGACUGGUAACUUUCAT 51 GAAAGUUACCAGUCUAUUATT 501 VEGFR-2 2686UAGAAGGUUGACCACAUUGAG 52 CAAUGUGGUCAACCUUCUATT 502 VEGFR-2 561UAGCUGAUCAUGUAGCUGGGA 53 CCAGCUACAUGAUCAGCUATT 503 VEGFR-2 525UUGCUGUCCCAGGAAAUUCTG 54 GAAUUUCCUGGGACAGCAATT 504 VEGFR-2 2277AUGAUUUCCAAGUUCGUCUTT 55 AGACGAACUUGGAAAUCAUTT 505 VEGFR-2 395UAAUGUACACGACUCCAUGTT 56 CAUGGAGUCGUGUACAUUATT 506 VEGFR-2 2410UUCAUCUGGAUCCAUGACGAT 57 CGUCAUGGAUCCAGAUGAATT 507 VEGFR-2 2007UGAUUCUCCAGGUUUCCUGTG 58 CAGGAAACCUGGAGAAUCATT 508 VEGFR-2 1323UAGACCGUACAUGUCAGCGTT 59 CGCUGACAUGUACGGUCUATT 509 VEGFR-2 3382UUCUGGUGUAGUAUAAUCAGG 60 UGAUUAUACUACACCAGAATT 510 VEGFR-2 3078UUUCGUGCCGCCAGGUCCCTG 61 GGGACCUGGCGGCACGAAATT 511 VEGFR-2 1432UUCUUCACAAGGGUAUGGGTT 62 CCCAUACCCUUGUGAAGAATT 512 VEGFR-2 1817UCAAUUUCCAAAGAGUAUCCA 63 GAUACUCUUUGGAAAUUGATT 513 VEGFR-2 688UAGUUCAAUUCCAUGAGACGG 64 GUCUCAUGGAAUUGAACUATT 514 VEGFR-2 2310AACAUGGCAAUCACCGCCGTG 65 CGGCGGUGAUUGCCAUGUUTT 515 VEGFR-2 2130UCCUUCAAUACAAUGCCUGAG 66 CAGGCAUUGUAUUGAAGGATT 516 VEGFR-2 799UACAAGUUUCUUAUGCUGATG 67 UCAGCAUAAGAAACUUGUATT 517 VEGFR-2 3523UGAUAUCGGAAGAACAAUGTA 68 CAUUGUUCUUCCGAUAUCATT 518 VEGFR-2 1843UGUGCUAUUAGAGAACAUGGT 69 CAUGUUCUCUAAUAGCACATT 519 VEGFR-2 2941UUCUACAUCACUGAGGGACTT 70 GUCCCUCAGUGAUGUAGAATT 520 VEGFR-2 2088UCUUUAAACCACAUGAUCUGT 71 AGAUCAUGUGGUUUAAAGATT 521 VEGFR-2 472UCUUGCACAAAGUGACACGTT 72 CGUGUCACUUUGUGCAAGATT 522 VEGFR-2 180UGAUUAUUGGGCCAAAGCCAG 73 GGCUUUGGCCCAAUAAUCATT 523 VEGFR-2 1568AUUUGUACAAAGCUGACACAT 74 GUGUCAGCUUUGUACAAAUTT 524 VEGFR-2 3141UAAAUAUCCCGGGCCAAGCCA 75 GCUUGGCCCGGGAUAUUUATT 525 VEGFR-2 3769AACCAUACCACUGUCCGUCTG 76 GACGGACAGUGGUAUGGUUTT 526 VEGFR-2 3920UGUCAUCGGAGUGAUAUCCGG 77 GGAUAUCACUCCGAUGACATT 527 VEGFR-2 1718UCUCAAACGUAGAUCUGUCTG 78 GACAGAUCUACGUUUGAGATT 528 VEGFR-2 2919UCCUCCACAAAUCCAGAGCTG 79 GCUCUGGAUUUGUGGAGGATT 529 VEGFR-2 324UAAAUGACCGAGGCCAAGUCA 80 ACUUGGCCUCGGUCAUUUATT 530 VEGFR-2 1050UAACCAAGGUACUUCGCAGGG 81 CUGCGAAGUACCUUGGUUATT 531 VEGFR-2 56UAGGCAAACCCACAGAGGCGG 82 GCCUCUGUGGGUUUGCCUATT 532 VEGFR-2 2453UGGCAUCAUAAGGCAGUCGTT 83 CGACUGCCUUAUGAUGCCATT 533 VEGFR-2 1303UUGAGUGGUGCCGUACUGGTA 84 CCAGUACGGCACCACUCAATT 534 VEGFR-2 1813UUUCCAAAGAGUAUCCAAGTT 85 CUUGGAUACUCUUUGGAAATT 535 VEGFR-2 2015UUGUCGUCUGAUUCUCCAGGT 86 CUGGAGAAUCAGACGACAATT 536 VEGFR-2 3088UAAGAGGAUAUUUCGUGCCGC 87 GGCACGAAAUAUCCUCUUATT 537 VEGFR-2 625UAUGUACAUAAUAGACUGGTA 88 CCAGUCUAUUAUGUACAUATT 538 VEGFR-2 800UUACAAGUUUCUUAUGCUGAT 89 CAGCAUAAGAAACUUGUAATT 539 VEGFR-2 811UAGGUCUCGGUUUACAAGUTT 90 ACUUGUAAACCGAGACCUATT 540 VEGFR-2 812UUAGGUCUCGGUUUACAAGTT 91 CUUGUAAACCGAGACCUAATT 541 VEGFR-2 3093UCCGAUAAGAGGAUAUUUCGT 92 GAAAUAUCCUCUUAUCGGATT 542 VEGFR-2 801UUUACAAGUUUCUUAUGCUGA 93 AGCAUAAGAAACUUGUAAATT 543 VEGFR-2 2009UCUGAUUCUCCAGGUUUCCTG 94 GGAAACCUGGAGAAUCAGATT 544 VEGFR-2 2127UUCAAUACAAUGCCUGAGUCT 95 ACUCAGGCAUUGUAUUGAATT 545 VEGFR-2 1585UUUGUUGACCGCUUCACAUTT 96 AUGUGAAGCGGUCAACAAATT 546 VEGFR-2 562AUAGCUGAUCAUGUAGCUGGG 97 CAGCUACAUGAUCAGCUAUTT 547 VEGFR-2 3906UAUCCGGACUGGUAGCCGCTT 98 GCGGCUACCAGUCCGGAUATT 548 VEGFR-2 1316UACAUGUCAGCGUUUGAGUGG 99 ACUCAAACGCUGACAUGUATT 549 VEGFR-2 3520UAUCGGAAGAACAAUGUAGTC 100 CUACAUUGUUCUUCCGAUATT 550 VEGFR-3 453UUCCUGUUGACCAAGAGCGTG 101 CGCUCUUGGUCAACAGGAATT 551 VEGFR-3 2694UUGAGCUCCGACAUCAGCGCG 102 CGCUGAUGUCGGAGCUCAATT 552 VEGFR-3 1689UUGGAUUCGAUGGUGAAGCCG 103 GCUUCACCAUCGAAUCCAATT 553 VEGFR-3 988UUCAUGCACAAUGACCUCGGT 104 CGAGGUCAUUGUGCAUGAATT 554 VEGFR-3 4374UUACCAAGGAAUAAUCGGCGG 105 GCCGAUUAUUCCUUGGUAATT 555 VEGFR-3 2142UCUUUGUACCACACGAUGCTG 106 GCAUCGUGUGGUACAAAGATT 556 VEGFR-3 1833UUGCAGUCGAGCAGAAGCGGG 107 CGCUUCUGCUCGACUGCAATT 557 VEGFR-3 3903UUCAGCUACCUGAAGCCGCTT 108 GCGGCUUCAGGUAGCUGAATT 558 VEGFR-3 3273UACACCUUGUCGAAGAUGCTT 109 GCAUCUUCGACAAGGUGUATT 559 VEGFR-3 1107UACCACUGGAACUCGGGCGGG 110 CGCCCGAGUUCCAGUGGUATT 560 VEGFR-3 336UAGCAGACGUAGCUGCCUGTG 111 CAGGCAGCUACGUCUGCUATT 561 VEGFR-3 2607UUGUGGAUGCCGAAAGCGGAG 112 CCGCUUUCGGCAUCCACAATT 562 VEGFR-3 1556UCACAGUCUUAUUCUUUCCCT 113 GGAAAGAAUAAGACUGUGATT 563 VEGFR-3 108UCCGUGAUGUUCAAGGUCGGG 114 CGACCUUGAACAUCACGGATT 564 VEGFR-3 1954AUAGUGGCCCUCGUGCUCGGG 115 CGAGCACGAGGGCCACUAUTT 565 VEGFR-3 2100AAGCACUGCAUCUCCAGCGAG 116 CGCUGGAGAUGCAGUGCUUTT 566 VEGFR-3 693UCAUAGAGCUCGUUGCCUGTG 117 CAGGCAACGAGCUCUAUGATT 567 VEGFR-3 2337AGGAUCACGAUCUCCAUGCTG 118 GCAUGGAGAUCGUGAUCCUTT 568 VEGFR-3 2054UCAAGUUCUGCGUGAGCCGAG 119 CGGCUCACGCAGAACUUGATT 569 VEGFR-3 860UCUGUUGGGAGCGUCGCUCGG 120 GAGCGACGCUCCCAACAGATT 570 VEGFR-3 2436UAGCCCGUCUUGAUGUCUGCG 121 CAGACAUCAAGACGGGCUATT 571 VEGFR-3 3759UUCAUCCUGGAGGAACCACGG 122 GUGGUUCCUCCAGGAUGAATT 572 VEGFR-3 288AACACCUUGCAGUAGGGCCTG 123 GGCCCUACUGCAAGGUGUUTT 573 VEGFR-3 1485UGCGUGGUCACCGCCCUCCAG 124 GGAGGGCGGUGACCACGCATT 574 VEGFR-3 2502UCGUAGGACAGGUAUUCGCAT 125 GCGAAUACCUGUCCUACGATT 575 VEGFR-3 925AUACGAGCCCAGGUCGUGCTG 126 GCACGACCUGGGCUCGUAUTT 576 VEGFR-3 426UUGUUGAUGAAUGGCUGCUCA 127 AGCAGCCAUUCAUCAACAATT 577 VEGFR-3 3189UAGAUGUCCCGGGCAAGGCCA 128 GCCUUGCCCGGGACAUCUATT 578 VEGFR-3 2274UUGACGCAGCCCUUGGGUCTG 129 GACCCAAGGGCUGCGUCAATT 579 VEGFR-3 2196UUCUGGUUGGAGUCCGCCAAG 130 UGGCGGACUCCAACCAGAATT 580 VEGFR-3 2019UGCACCGACAGGUACUUCUTG 131 AGAAGUACCUGUCGGUGCATT 581 VEGFR-3 360AUGCGUGCCUUGAUGUACUTG 132 AGUACAUCAAGGCACGCAUTT 582 VEGFR-3 1755UACUUGUAGCUGUCGGCUUGG 133 AAGCCGACAGCUACAAGUATT 583 VEGFR-3 3037UUCCAUGGUCAGCGGGCUCAG 134 GAGCCCGCUGACCAUGGAATT 584 VEGFR-3 1018UUUGAGCCACUCGACGCUGAT 135 CAGCGUCGAGUGGCUCAAATT 585 VEGFR-3 1684UUCGAUGGUGAAGCCGUCGGG 136 CGACGGCUUCACCAUCGAATT 586 VEGFR-3 4373UACCAAGGAAUAAUCGGCGGG 137 CGCCGAUUAUUCCUUGGUATT 587 VEGFR-3 987UCAUGCACAAUGACCUCGGTG 138 CCGAGGUCAUUGUGCAUGATT 588 VEGFR-3 3267UUGUCGAAGAUGCUUUCAGGG 139 CUGAAAGCAUCUUCGACAATT 589 VEGFR-3 4387UGUAUUACUCAUAUUACCAAG 140 UGGUAAUAUGAGUAAUACATT 590 VEGFR-3 3883UUCUUGUCUAUGCCUGCUCTC 141 GAGCAGGCAUAGACAAGAATT 591 VEGFR-3 4376UAUUACCAAGGAAUAAUCGGC 142 CGAUUAUUCCUUGGUAAUATT 592 VEGFR-3 2140UUUGUACCACACGAUGCUGGG 143 CAGCAUCGUGUGGUACAAATT 593 VEGFR-3 978AUGACCUCGGUGCUCUCCCGA 144 GGGAGAGCACCGAGGUCAUTT 594 VEGFR-3 2427UUGAUGUCUGCGUGGGCCGGC 145 CGGCCCACGCAGACAUCAATT 595 VEGFR-3 1109UGUACCACUGGAACUCGGGCG 146 CCCGAGUUCCAGUGGUACATT 596 VEGFR-3 319UGUGUCGUUGGCAUGUACCTC 147 GGUACAUGCCAACGACACATT 597 VEGFR-3 1843AUGCACGUUCUUGCAGUCGAG 148 CGACUGCAAGAACGUGCAUTT 598 VEGFR-3 317UGUCGUUGGCAUGUACCUCGT 149 GAGGUACAUGCCAACGACATT 599 VEGFR-3 700CUGGAUGUCAUAGAGCUCGTT 150 CGAGCUCUAUGACAUCCAGTT 600 Tie-2 1223UAAGCUUACAAUCUGGCCCGT 151 GGGCCAGAUUGUAAGCUUATT 601 (TEK) Tie-2 2350UAUCUUCACAUCAACGUGCTG 152 GCACGUUGAUGUGAAGAUATT 602 (TEK) Tie-2 706UAUGUUCACGUUAUCUCCCTT 153 GGGAGAUAACGUGAACAUATT 603 (TEK) Tie-2 3561UUUAAGGACACCAAUAUCUGG 154 AGAUAUUGGUGUCCUUAAATT 604 (TEK) Tie-2 2763UGAAAUUUGAUGUCAUUCCAG 155 GGAAUGACAUCAAAUUUCATT 605 (TEK) Tie-2 174UUGUUUACAAGUUAGAGGCAA 156 GCCUCUAACUUGUAAACAATT 606 (TEK) Tie-2 1183UUCAUUGCACUGCAGACCCTT 157 GGGUCUGCAGUGCAAUGAATT 607 (TEK) Tie-2 805UAGAAUAUCAGGUACUUCATG 158 UGAAGUACCUGAUAUUCUATT 608 (TEK) Tie-2 2601UUCAAUUGCAAUAUGAUCAGA 159 UGAUCAUAUUGCAAUUGAATT 609 (TEK) Tie-2 2277UAGCCAUCCAAUAUUGUCCAA 160 GGACAAUAUUGGAUGGCUATT 610 (TEK) Tie-2 1366UACUUCUAUAUGAUCUGGCAA 161 GCCAGAUCAUAUAGAAGUATT 611 (TEK) Tie-2 32UUUGGUAUCAGCAGGGCUGGG 162 CAGCCCUGCUGAUACCAAATT 612 (TEK) Tie-2 4085UGUACUAUCAGGGUCAUUGTT 163 CAAUGACCCUGAUAGUACATT 613 (TEK) Tie-2 3881UUCUGAUUUCAGCCCAUUCTT 164 GAAUGGGCUGAAAUCAGAATT 614 (TEK) Tie-2 646UUGUUGACGCAUCUUCAUGGT 165 CAUGAAGAUGCGUCAACAATT 615 (TEK) Tie-2 4021AUAGCAUUCAACAUAAAGGTA 166 CCUUUAUGUUGAAUGCUAUTT 616 (TEK) Tie-2 209UUUGUGACUUUCCAUUAGCAT 167 GCUAAUGGAAAGUCACAAATT 617 (TEK) Tie-2 4223UAAAUGAAACGGGACUGGCTG 168 GCCAGUCCCGUUUCAUUUATT 618 (TEK) Tie-2 3961UACUAAUUGUACUCACGCCTT 169 GGCGUGAGUACAAUUAGUATT 619 (TEK) Tie-2 1771UUGAAUAUGUUGCCAAGCCTC 170 GGCUUGGCAACAUAUUCAATT 620 (TEK) Tie-2 3909UUAUUGCAUAUGAAACCACAA 171 GUGGUUUCAUAUGCAAUAATT 621 (TEK) Tie-2 3606UAAAGCGUGGUAUUCACGUAG 172 ACGUGAAUACCACGCUUUATT 622 (TEK) Tie-2 477AUUAAGGCUUCAAAGUCCCTT 173 GGGACUUUGAAGCCUUAAUTT 623 (TEK) Tie-2 3421UUCUGCACAAGUCAUCCCGCA 174 CGGGAUGACUUGUGCAGAATT 624 (TEK) Tie-2 2730UAAAUUGUAGGAUCUGGGUTG 175 ACCCAGAUCCUACAAUUUATT 625 (TEK) Tie-2 1800UAGUUGAGUGUAACAAUCUCA 176 AGAUUGUUACACUCAACUATT 626 (TEK) Tie-2 3385UAAGCUAACAAUCUCCCAUAG 177 AUGGGAGAUUGUUAGCUUATT 627 (TEK) Tie-2 1692UAAGGCUCAGAGCUGAUGUTG 178 ACAUCAGCUCUGAGCCUUATT 628 (TEK) Tie-2 1657AUGUCCAGUGUCAAUCACGTT 179 CGUGAUUGACACUGGACAUTT 629 (TEK) Tie-2 3665UUCUGUCCUAGGCCGCUUCTT 180 GAAGCGGCCUAGGACAGAATT 630 (TEK) Tie-2 2091UUAAGUAGCACCGAAGUCAAG 181 UGACUUCGGUGCUACUUAATT 631 (TEK) Tie-2 2827UAACCCAUCCUUCUUGAUGCG 182 CAUCAAGAAGGAUGGGUUATT 632 (TEK)  Tie-2 1979UUGGUUGCCAGGUCAAAUUTA 183 AAUUUGACCUGGCAACCAATT 633 (TEK) Tie-2 67UAGAUUAGGAUGGGAAAGGCT 184 CCUUUCCCAUCCUAAUCUATT 634 (TEK) Tie-2 3459UUCUCCAGUCUGUAGCCCUGG 185 AGGGCUACAGACUGGAGAATT 635 (TEK) Tie-2 2764UUGAAAUUUGAUGUCAUUCCA 186 GAAUGACAUCAAAUUUCAATT 636 (TEK) Tie-2 3560UUAAGGACACCAAUAUCUGGG 187 CAGAUAUUGGUGUCCUUAATT 637 (TEK) Tie-2 715UUUGAAAGAUAUGUUCACGTT 188 CGUGAACAUAUCUUUCAAATT 638 (TEK) Tie-2 1368UUUACUUCUAUAUGAUCUGGC 189 CAGAUCAUAUAGAAGUAAATT 639 (TEK) Tie-2 2351UUAUCUUCACAUCAACGUGCT 190 CACGUUGAUGUGAAGAUAATT 640 (TEK) Tie-2 205UGACUUUCCAUUAGCAUCGTC 191 CGAUGCUAAUGGAAAGUCATT 641 (TEK) Tie-2 3957AAUUGUACUCACGCCUUCCTA 192 GGAAGGCGUGAGUACAAUUTT 642 (TEK) Tie-2 3962AUACUAAUUGUACUCACGCCT 193 GCGUGAGUACAAUUAGUAUTT 643 (TEK) Tie-2 2352UUUAUCUUCACAUCAACGUGC 194 ACGUUGAUGUGAAGAUAAATT 644 (TEK) Tie-2 3963UAUACUAAUUGUACUCACGCC 195 CGUGAGUACAAUUAGUAUATT 645 (TEK) Tie-2 1777UGUCACUUGAAUAUGUUGCCA 196 GCAACAUAUUCAAGUGACATT 646 (TEK) Tie-2 3388UCCUAAGCUAACAAUCUCCCA 197 GGAGAUUGUUAGCUUAGGATT 647 (TEK) Tie-2 636AUCUUCAUGGUUCGUAUCCTG 198 GGAUACGAACCAUGAAGAUTT 648 (TEK) Tie-2 74UCCUUUGUAGAUUAGGAUGGG 199 CAUCCUAAUCUACAAAGGATT 649 (TEK) Tie-2 707AUAUGUUCACGUUAUCUCCCT 200 GGAGAUAACGUGAACAUAUTT 650 (TEK) bFGFR 3814UAAAUCUCUGGUAACGACCCT 201 GGUCGUUACCAGAGAUUUATT 651 bFGFR 1478UUACACAUGAACUCCACGUTG 202 ACGUGGAGUUCAUGUGUAATT 652 bFGFR 3773UAUACUCAGAUUUAUCAACTT 203 GUUGAUAAAUCUGAGUAUATT 653 bFGFR 715UAGCGGUGCAGAGUGUGGCTG 204 GCCACACUCUGCACCGCUATT 654 bFGFR 575UUCAAACUGACCCUCGCUCGG 205 GAGCGAGGGUCAGUUUGAATT 655 bFGFR 646UUCUGCAGUUAGAGGUUGGTG 206 CCAACCUCUAACUGCAGAATT 656 bFGFR 3625AUCGGAAUUAAUAAGCCACTG 207 GUGGCUUAUUAAUUCCGAUTT 657 bFGFR 2318UACAAGGGACCAUCCUGCGTG 208 CGCAGGAUGGUCCCUUGUATT 658 bFGFR 1439UUGUUGGCGGGCAACCCUGCT 209 CAGGGUUGCCCGCCAACAATT 659 bFGFR 3860AUAGCAACUGAUGCCUCCCAG 210 GGGAGGCAUCAGUUGCUAUTT 660 bFGFR 3163UGAGGGUUACAGCUGACGGTG 211 CCGUCAGCUGUAACCCUCATT 661 bFGFR 2600UCGAUGUGGUGAAUGUCCCGT 212 GGGACAUUCACCACAUCGATT 662 bFGFR 2513UCUCGGUGUAUGCACUUCUTG 213 AGAAGUGCAUACACCGAGATT 663 bFGFR 2214UUUCUCUGUUGCGUCCGACTT 214 GUCGGACGCAACAGAGAAATT 664 bFGFR 1346UUCUCCACAAUGCAGGUGUAG 215 ACACCUGCAUUGUGGAGAATT 665 bFGFR 1556UUGUCUGGGCCAAUCUUGCTC 216 GCAAGAUUGGCCCAGACAATT 666 bFGFR 2671UCCGGUCAAAUAAUGCCUCGG 217 GAGGCAUUAUUUGACCGGATT 667 bFGFR 3105UUUGAGUCCGCCAUUGGCAAG 218 UGCCAAUGGCGGACUCAAATT 668 bFGFR 2091UUUGCCUAAGACCAGUCUGTC 219 CAGACUGGUCUUAGGCAAATT 669 bFGFR 1590UCCAGCAGUCUUCAAGAUCTG 220 GAUCUUGAAGACUGCUGGATT 670 bFGFR 1689UCCGAUAGAGUUACCCGCCAA 221 GGCGGGUAACUCUAUCGGATT 671 bFGFR 1319UUGUCAGAGGGCACCACAGAG 222 CUGUGGUGCCCUCUGACAATT 672 bFGFR 2342UUGGAGGCAUACUCCACGATG 223 UCGUGGAGUAUGCCUCCAATT 673 bFGFR 107UCUCGGUCCCGACCGGACGTG 224 CGUCCGGUCGGGACCGAGATT 674 bFGFR 3662UCUGGUACCAGGCAUUUGGTC 225 CCAAAUGCCUGGUACCAGATT 675 bFGFR 2150UUGUCCAGCCCGAUAGCCUCT 226 AGGCUAUCGGGCUGGACAATT 676 bFGFR 1517UUUAGCCACUGGAUGUGCGGC 227 CGCACAUCCAGUGGCUAAATT 677 bFGFR 1264UGUAGCCUCCAAUUCUGUGGT 228 CACAGAAUUGGAGGCUACATT 678 bFGFR 3576UUCAAUCGUGGCUCGAAGCAC 229 GCUUCGAGCCACGAUUGAATT 679 bFGFR 613AUCUCCAUGGAUACUCCACAG 230 GUGGAGUAUCCAUGGAGAUTT 680 bFGFR 1221UUUCAACCAGCGCAGUGUGGG 231 CACACUGCGCUGGUUGAAATT 681 bFGFR 3004UAGAGCUCCGGGUGUCGGGAA 232 CCCGACACCCGGAGCUCUATT 682 bFGFR 3825UUACCGAUGGGUAAAUCUCTG 233 GAGAUUUACCCAUCGGUAATT 683 bFGFR 3813AAAUCUCUGGUAACGACCCTT 234 GGGUCGUUACCAGAGAUUUTT 684 bFGFR 3861UAUAGCAACUGAUGCCUCCCA 235 GGAGGCAUCAGUUGCUAUATT 685 bFGFR 576UUUCAAACUGACCCUCGCUCG 236 AGCGAGGGUCAGUUUGAAATT 686 bFGFR 3772AUACUCAGAUUUAUCAACUTT 237 AGUUGAUAAAUCUGAGUAUTT 687 bFGFR 3824UACCGAUGGGUAAAUCUCUGG 238 AGAGAUUUACCCAUCGGUATT 688 bFGFR 2319AUACAAGGGACCAUCCUGCGT 239 GCAGGAUGGUCCCUUGUAUTT 689 bFGFR 3771UACUCAGAUUUAUCAACUUTG 240 AAGUUGAUAAAUCUGAGUATT 690 bFGFR 2511UCGGUGUAUGCACUUCUUGGA 241 CAAGAAGUGCAUACACCGATT 691 bFGFR 2333UACUCCACGAUGACAUACAAG 242 UGUAUGUCAUCGUGGAGUATT 692 bFGFR 3624UCGGAAUUAAUAAGCCACUGG 243 AGUGGCUUAUUAAUUCCGATT 693 bFGFR 1304ACAGAGUCCAUUAUGAUGCTC 244 GCAUCAUAAUGGACUCUGUTT 694 bFGFR 1608UUUGUCGGUGGUAUUAACUCC  245 AGUUAAUACCACCGACAAATT 695 bFGFR 1301GAGUCCAUUAUGAUGCUCCAG 246 GGAGCAUCAUAAUGGACUCTT 696 bFGFR 3626UAUCGGAAUUAAUAAGCCACT 247 UGGCUUAUUAAUUCCGAUATT 697 bFGFR 2672AUCCGGUCAAAUAAUGCCUCG 248 AGGCAUUAUUUGACCGGAUTT 698 bFGFR 2213UUCUCUGUUGCGUCCGACUTC 249 AGUCGGACGCAACAGAGAATT 699 bFGFR 2597AUGUGGUGAAUGUCCCGUGCG 250 CACGGGACAUUCACCACAUTT 700 IL8RA 1971UUUAUUAGGAACAUCUGCCTG 251 GGCAGAUGUUCCUAAUAAATT 701 IL8RA 75UUGAUCUAACUGAAGCACCGG 252 GGUGCUUCAGUUAGAUCAATT 702 IL8RA 645AUUGUUUGGAUGGUAAGCCTG 253 GGCUUACCAUCCAAACAAUTT 703 IL8RA 1431UAAUUAGCCAGUUAGUGGGTT 254 CCCACUAACUGGCUAAUUATT 704 IL8RA 1378UUCGUUUCCAUGGAGGUGCAA 255 GCACCUCCAUGGAAACGAATT 705 IL8RA 1470UCAUCUAAUGUCAGAUUCGGG 256 CGAAUCUGACAUUAGAUGATT 706 IL8RA 218UACUUGUUGAGUGUCUCAGTT 257 CUGAGACACUCAACAAGUATT 707 IL8RA 1101AUGACGUGCCAAGAACUCCTT 258 GGAGUUCUUGGCACGUCAUTT 708 IL8RA 677UUUCCCAGGACCUCAUAGCAA 259 GCUAUGAGGUCCUGGGAAATT 709 IL8RA 1178AAGAGAUAUUCCUUCAUCGAT 260 CGAUGAAGGAAUAUCUCUUTT 710 IL8RA 1543UUGAGGAGAUGCUCCUGUGAG 261 CACAGGAGCAUCUCCUCAATT 711 IL8RA 1783UCUUGUGGCAUAGAUCUGGCT 262 CCAGAUCUAUGCCACAAGATT 712 IL8RA 1249AUAGUGCCUGUCCAGAGCCAG 263 GGCUCUGGACAGGCACUAUTT 713 IL8RA 1520UCAACGAGAGCAUCCAGCCCT 264 GGCUGGAUGCUCUCGUUGATT 714 IL8RA 1068AUGCAUAGCCAGGAUCUUGAG 265 CAAGAUCCUGGCUAUGCAUTT 715 IL8RA 1347UUGGAGGUACCUCAACAGCTC 266 GCUGUUGAGGUACCUCCAATT 716 IL8RA 1208UCAGGGUGUUGGUUAUUCUTT 267 AGAAUAACCAACACCCUGATT 717 IL8RA 117AUCUGUAAUAUUUGACAUGTC 268 CAUGUCAAAUAUUACAGAUTT 718 IL8RA 1862UGCUUGUCUCGUUCCACUUGG 269 AAGUGGAACGAGACAAGCATT 719 IL8RA 1153UUCAGAGGUUGGAAGAGACAT 270 GUCUCUUCCAACCUCUGAATT 720 IL8RA 640UUGGAUGGUAAGCCUGGCGGA 271 CGCCAGGCUUACCAUCCAATT 721 IL8RA 1411UAAAGAUGUGACGUUCAACGG 272 GUUGAACGUCACAUCUUUATT 722 IL8RA 71UCUAACUGAAGCACCGGCCAG 273 GGCCGGUGCUUCAGUUAGATT 723 IL8RA 1397UCAACGGGAAUGAUGGUGCTT 274 GCACCAUCAUUCCCGUUGATT 724 IL8RA 644UUGUUUGGAUGGUAAGCCUGG 275 AGGCUUACCAUCCAAACAATT 725 IL8RA 641UUUGGAUGGUAAGCCUGGCGG 276 GCCAGGCUUACCAUCCAAATT 726 IL8RA 76UUUGAUCUAACUGAAGCACCG 277 GUGCUUCAGUUAGAUCAAATT 727 IL8RA 1398UUCAACGGGAAUGAUGGUGCT 278 CACCAUCAUUCCCGUUGAATT 728 IL8RA 1381UGCUUCGUUUCCAUGGAGGTG 279 CCUCCAUGGAAACGAAGCATT 729 IL8RA 1769UCUGGCUUCCAAACCCUCUTT 280 AGAGGGUUUGGAAGCCAGATT 730 IL8RA 1435AUGCUAAUUAGCCAGUUAGTG 281 CUAACUGGCUAAUUAGCAUTT 731 IL8RA 1175AGAUAUUCCUUCAUCGAUGGT 282 CAUCGAUGAAGGAAUAUCUTT 732 IL8RA 1970UUAUUAGGAACAUCUGCCUGC 283 AGGCAGAUGUUCCUAAUAATT 733 IL8RA 1432CUAAUUAGCCAGUUAGUGGGT 284 CCACUAACUGGCUAAUUAGTT 734 IL8RA 74UGAUCUAACUGAAGCACCGGC 285 CGGUGCUUCAGUUAGAUCATT 735 IL8RA 646AAUUGUUUGGAUGGUAAGCCT 286 GCUUACCAUCCAAACAAUUTT 736 IL8RA 639UGGAUGGUAAGCCUGGCGGAA 287 CCGCCAGGCUUACCAUCCATT 737 IL8RA 1082UUGCUGACCAGGCCAUGCATA 288 UGCAUGGCCUGGUCAGCAATT 738 IL8RA 1770AUCUGGCUUCCAAACCCUCTT 289 GAGGGUUUGGAAGCCAGAUTT 739 IL8RA 81AAUGGUUUGAUCUAACUGAAG 290 UCAGUUAGAUCAAACCAUUTT 740 IL8RA 1372UCCAUGGAGGUGCAAAGGCCG 291 GCCUUUGCACCUCCAUGGATT 741 IL8RA 1388AUGAUGGUGCUUCGUUUCCAT 292 GGAAACGAAGCACCAUCAUTT 742 IL8RA 643UGUUUGGAUGGUAAGCCUGGC 293 CAGGCUUACCAUCCAAACATT 743 IL8RA 1784UUCUUGUGGCAUAGAUCUGGC 294 CAGAUCUAUGCCACAAGAATT 744 IL8RA 1524AGGGUCAACGAGAGCAUCCAG 295 GGAUGCUCUCGUUGACCCUTT 745 IL8RA 237AUAGGCGAUGAUCACAACATA 296 UGUUGUGAUCAUCGCCUAUTT 746 IL8RA 219AUACUUGUUGAGUGUCUCAGT 297 UGAGACACUCAACAAGUAUTT 747 IL8RA 1389AAUGAUGGUGCUUCGUUUCCA 298 GAAACGAAGCACCAUCAUUTT 748 IL8RA 1972CUUUAUUAGGAACAUCUGCCT 299 GCAGAUGUUCCUAAUAAAGTT 749 IL8RA 1115UAGGAGGUAACACGAUGACGT 300 GUCAUCGUGUUACCUCCUATT 750 IL8RB 2648UUAAGUGUCAAUUUAGUGGCA 301 CCACUAAAUUGACACUUAATT 751 IL8RB 2184UUUCUUGUGGGUCAAUUCCTA 302 GGAAUUGACCCACAAGAAATT 752 IL8RB 2250UUGGGUCUUGUGAAUAAGCTG 303 GCUUAUUCACAAGACCCAATT 753 IL8RB 1746UUCACUUCUUAGAACAUAGAG 304 CUAUGUUCUAAGAAGUGAATT 754 IL8RB 960UUGGAUGAGUAGACGGUCCTT 305 GGACCGUCUACUCAUCCAATT 755 IL8RB 454AUUACUAAGAUCUUCACCUTT 306 AGGUGAAGAUCUUAGUAAUTT 756 IL8RB 2750UUGGUUUAAUCAGCCUUGGTG 307 CCAAGGCUGAUUAAACCAATT 757 IL8RB 2604AUCACUACUGUUUAUCUGCAG 308 GCAGAUAAACAGUAGUGAUTT 758 IL8RB 1026AUCCGUAACAGCAUCCGCCAG 309 GGCGGAUGCUGUUACGGAUTT 759 IL8RB 1384AUGUAUAGCUAGAAUCUUGAG 310 CAAGAUUCUAGCUAUACAUTT 760 IL8RB 1149AAGAUGACCCGCAUGGCCCGG 311 GGGCCAUGCGGGUCAUCUUTT 761 IL8RB 2464UCUCAGUACCUCAUGUAGGTG 312 CCUACAUGAGGUACUGAGATT 762 IL8RB 877UUUGACCAAGUAGCGCUUCTG 313 GAAGCGCUACUUGGUCAAATT 763 IL8RB 2324UUCGUUAGGUACAUAUCACAT 314 GUGAUAUGUACCUAACGAATT 764 IL8RB 2360AUGAGUACUUCAUUCCUCUTT 315 AGAGGAAUGAAGUACUCAUTT 765 IL8RB 265UUGGGUGGUAGUCAGAGCUGT 316 AGCUCUGACUACCACCCAATT 766 IL8RB 1642UUUCUAAACCAUGCAAGGGAA 317 CCCUUGCAUGGUUUAGAAATT 767 IL8RB 2146UCAUGUGUUAAUUCUAUGUCT 318 ACAUAGAAUUAACACAUGATT 768 IL8RB 2627UUAAGUCACAUUGCGGUACAA 319 GUACCGCAAUGUGACUUAATT 769 IL8RB 1000UGUAUUGUUGCCCAUGUCCTC 320 GGACAUGGGCAACAAUACATT 770 IL8RB 315UGACCUGCUGUUAUUGGAGTG 321 CUCCAAUAACAGCAGGUCATT 771 IL8RB 2774AAAUAUAGGCAGGUGGUUCTA 322 GAACCACCUGCCUAUAUUUTT 772 IL8RB 219ACCUUGACGAUGAAACUUCTG 323 GAAGUUUCAUCGUCAAGGUTT 773 IL8RB 2389UUUCAAGGUUCGUCCGUGUTG 324 ACACGGACGAACCUUGAAATT 774 IL8RB 385UGAGGUAAACUUAAAUCCUGA 325 AGGAUUUAAGUUUACCUCATT 775 IL8RB 1347UUCUGGCCAAUGAAGGCGUAG 326 ACGCCUUCAUUGGCCAGAATT 776 IL8RB 2649UUUAAGUGUCAAUUUAGUGGC 327 CACUAAAUUGACACUUAAATT 777 IL8RB 1737UAGAACAUAGAGUGCCAUGGG 328 CAUGGCACUCUAUGUUCUATT 778 IL8RB 455AAUUACUAAGAUCUUCACCTT 329 GGUGAAGAUCUUAGUAAUUTT 779 IL8RB 965UAACAUUGGAUGAGUAGACGG 330 GUCUACUCAUCCAAUGUUATT 780 IL8RB 1740UCUUAGAACAUAGAGUGCCAT 331 GGCACUCUAUGUUCUAAGATT 781 IL8RB 2632UGGCAUUAAGUCACAUUGCGG 332 GCAAUGUGACUUAAUGCCATT 782 IL8RB 2755UAGCCUUGGUUUAAUCAGCCT 333 GCUGAUUAAACCAAGGCUATT 783 IL8RB 2183UUCUUGUGGGUCAAUUCCUAT 334 AGGAAUUGACCCACAAGAATT 784 IL8RB 2605UAUCACUACUGUUUAUCUGCA 335 CAGAUAAACAGUAGUGAUATT 785 IL8RB 2340UCAGGCUGAAGGAUACUUCGT 336 GAAGUAUCCUUCAGCCUGATT 786 IL8RB 2143UGUGUUAAUUCUAUGUCUGAA 337 CAGACAUAGAAUUAACACATT 787 IL8RB 998UAUUGUUGCCCAUGUCCUCAT 338 GAGGACAUGGGCAACAAUATT 788 IL8RB 2180UUGUGGGUCAAUUCCUAUAAG 339 UAUAGGAAUUGACCCACAATT 789 IL8RB 2185AUUUCUUGUGGGUCAAUUCCT 340 GAAUUGACCCACAAGAAAUTT 790 IL8RB 307UGUUAUUGGAGUGGCCACCGA 341 GGUGGCCACUCCAAUAACATT 791 IL8RB 2481UCUGUAAAUUUGUUCACUCTC 342 GAGUGAACAAAUUUACAGATT 792 IL8RB 2617UUGCGGUACAACUAUCACUAC 343 AGUGAUAGUUGUACCGCAATT 793 IL8RB 956AUGAGUAGACGGUCCUUCGGA 344 CGAAGGACCGUCUACUCAUTT 794 IL8RB 456UAAUUACUAAGAUCUUCACCT 345 GUGAAGAUCUUAGUAAUUATT 795 IL8RB 226UGAAACAACCUUGACGAUGAA 346 CAUCGUCAAGGUUGUUUCATT 796 IL8RB 1394UGAUCAAGCCAUGUAUAGCTA 347 GCUAUACAUGGCUUGAUCATT 797 IL8RB 458UGUAAUUACUAAGAUCUUCAC 348 GAAGAUCUUAGUAAUUACATT 798 IL8RB 881UGAAUUUGACCAAGUAGCGCT 349 CGCUACUUGGUCAAAUUCATT 799 IL8RB 2327UACUUCGUUAGGUACAUAUCA 350 AUAUGUACCUAACGAAGUATT 800 Fas 109UGUAGUAACAGUCUUCCUCAA 351 GAGGAAGACUGUUACUACATT 801 Fas 41UGGACGAUAAUCUAGCAACAG 352 GUUGCUAGAUUAUCGUCCATT 802 Fas 161UAUGGCAGAAUUGGCCAUCAT 353 GAUGGCCAAUUCUGCCAUATT 803 Fas 182UUUCACCUGGAGGACAGGGCT 354 CCCUGUCCUCCAGGUGAAATT 804 Fas 62UCACUUGGGCAUUAACACUTT 355 AGUGUUAAUGCCCAAGUGATT 805 Fas 377ACUUCCUCUUUGCACUUGGTG 356 CCAAGUGCAAAGAGGAAGUTT 806 Fas 349UGAGUGUGCAUUCCUUGAUGA 357 AUCAAGGAAUGCACACUCATT 807 Fas 245UCCCUUCUUGGCAGGGCACGC 358 GUGCCCUGCCAAGAAGGGATT 808 Fas 205GACUGUGCAGUCCCUAGCUTT 359 AGCUAGGGACUGCACAGUCTT 809 Fas 145AUCAUGAUGCAGGCCUUCCAA 360 GGAAGGCCUGCAUCAUGAUTT 810 Fas 123UUCUGAGUCUCAACUGUAGTA 361 CUACAGUUGAGACUCAGAATT 811 Fas 34UAAUCUAGCAACAGACGUAAG 362 UACGUCUGUUGCUAGAUUATT 812 Fas 114UCAACUGUAGUAACAGUCUTC 363 AGACUGUUACUACAGUUGATT 813 Fas 115CUCAACUGUAGUAACAGUCTT 364 GACUGUUACUACAGUUGAGTT 814 Fas 28AGCAACAGACGUAAGAACCAG 365 GGUUCUUACGUCUGUUGCUTT 815 Fas 122UCUGAGUCUCAACUGUAGUAA 366 ACUACAGUUGAGACUCAGATT 816 Fas 186UUCCUUUCACCUGGAGGACAG 367 GUCCUCCAGGUGAAAGGAATT 817 Fas 42UUGGACGAUAAUCUAGCAACA 368 UUGCUAGAUUAUCGUCCAATT 818 Fas 111ACUGUAGUAACAGUCUUCCTC 369 GGAAGACUGUUACUACAGUTT 819 Fas 144UCAUGAUGCAGGCCUUCCAAG 370 UGGAAGGCCUGCAUCAUGATT 820 Fas 92UCAAUUCCAAUCCCUUGGAGT 371 UCCAAGGGAUUGGAAUUGATT 821 Fas 201GUGCAGUCCCUAGCUUUCCTT 372 GGAAAGCUAGGGACUGCACTT 822 Fas 128CCAAGUUCUGAGUCUCAACTG 373 GUUGAGACUCAGAACUUGGTT 823 Fas 36GAUAAUCUAGCAACAGACGTA 374 CGUCUGUUGCUAGAUUAUCTT 824 Fas 162UUAUGGCAGAAUUGGCCAUCA 375 AUGGCCAAUUCUGCCAUAATT 825 Fas 127CAAGUUCUGAGUCUCAACUGT 376 AGUUGAGACUCAGAACUUGTT 826 Fas 202UGUGCAGUCCCUAGCUUUCCT 377 GAAAGCUAGGGACUGCACATT 827 Fas 82UCCCUUGGAGUUGAUGUCAGT 378 UGACAUCAACUCCAAGGGATT 828 Fas 160AUGGCAGAAUUGGCCAUCATG 379 UGAUGGCCAAUUCUGCCAUTT 829 Fas 150UGGCCAUCAUGAUGCAGGCCT 380 GCCUGCAUCAUGAUGGCCATT 830 Fas 63GUCACUUGGGCAUUAACACTT 381 GUGUUAAUGCCCAAGUGACTT 831 Fas 164GCUUAUGGCAGAAUUGGCCAT 382 GGCCAAUUCUGCCAUAAGCTT 832 Fas 37CGAUAAUCUAGCAACAGACGT 383 GUCUGUUGCUAGAUUAUCGTT 833 Fas 116UCUCAACUGUAGUAACAGUCT 384 ACUGUUACUACAGUUGAGATT 834 Fas 32AUCUAGCAACAGACGUAAGAA 385 CUUACGUCUGUUGCUAGAUTT 835 Fas 64AGUCACUUGGGCAUUAACACT 386 UGUUAAUGCCCAAGUGACUTT 836 Fas 167AGGGCUUAUGGCAGAAUUGGC 387 CAAUUCUGCCAUAAGCCCUTT 837 Fas 120UGAGUCUCAACUGUAGUAACA 388 UUACUACAGUUGAGACUCATT 838 Fas 125AGUUCUGAGUCUCAACUGUAG 389 ACAGUUGAGACUCAGAACUTT 839 Fas 43UUUGGACGAUAAUCUAGCAAC 390 UGCUAGAUUAUCGUCCAAATT 840 Fas 94CCUCAAUUCCAAUCCCUUGGA 391 CAAGGGAUUGGAAUUGAGGTT 841 Fas 159UGGCAGAAUUGGCCAUCAUGA 392 AUGAUGGCCAAUUCUGCCATT 842 Fas 110CUGUAGUAACAGUCUUCCUCA 393 AGGAAGACUGUUACUACAGTT 843 Fas 31UCUAGCAACAGACGUAAGAAC 394 UCUUACGUCUGUUGCUAGATT 844 Fas 38ACGAUAAUCUAGCAACAGACG 395 UCUGUUGCUAGAUUAUCGUTT 845 Fas 118AGUCUCAACUGUAGUAACAGT 396 UGUUACUACAGUUGAGACUTT 846 Fas 169ACAGGGCUUAUGGCAGAAUTG 397 AUUCUGCCAUAAGCCCUGUTT 847 Fas 33AAUCUAGCAACAGACGUAAGA 398 UUACGUCUGUUGCUAGAUUTT 848 Fas 163CUUAUGGCAGAAUUGGCCATC 399 UGGCCAAUUCUGCCAUAAGTT 849 Fas 233AGGGCACGCAGUCUGGUUCAT 400 GAACCAGACUGCGUGCCCUTT 850 IGF2R 6340UUUGUCACCUAUGACACCCAG 401 GGGUGUCAUAGGUGACAAATT 851 IGF2R 2936UUAUAGAGCAAGCCUGGUCTG 402 GACCAGGCUUGCUCUAUAATT 852 IGF2R 1331UCUGAUUGUGGUAUCUUCCTG 403 GGAAGAUACCACAAUCAGATT 853 IGF2R 4491UAUUUCAGGACAAUUAUGCCA 404 GCAUAAUUGUCCUGAAAUATT 854 IGF2R 2562UUAAUGUAGUAUUUCCUCCAC 405 GGAGGAAAUACUACAUUAATT 855 IGF2R 1456UUUCCCAUCGUUACCUGCGGT 406 CGCAGGUAACGAUGGGAAATT 856 IGF2R 2253UAGUUCAGUUGGAUCAUCCCA 407 GGAUGAUCCAACUGAACUATT 857 IGF2R 3570UUGCCUUCUGACACUAAGCAA 408 GCUUAGUGUCAGAAGGCAATT 858 IGF2R 2274UUAUAGGGUGUGCCGCCUCTG 409 GAGGCGGCACACCCUAUAATT 859 IGF2R 1197UUUCCAUCUGAAAUAUAGGAT 410 CCUAUAUUUCAGAUGGAAATT 860 IGF2R 897UUGCGCACCAGCUUCAGUCCG 411 GACUGAAGCUGGUGCGCAATT 861 IGF2R 5205UUGAUGUAGAAAUCAGGGUTG 412 ACCCUGAUUUCUACAUCAATT 862 IGF2R 8904UUCUCAGCAAUAGAACACCAG 413 GGUGUUCUAUUGCUGAGAATT 863 IGF2R 8604UAAGGCUUCUUAUAGGUCGAA 414 CGACCUAUAAGAAGCCUUATT 864 IGF2R 3629UCAAAGAUCCAUUCGCCGCGG 415 GCGGCGAAUGGAUCUUUGATT 865 IGF2R 4344UUGAUGAGGUAGUGCUCCGGG 416 CGGAGCACUACCUCAUCAATT 866 IGF2R 1419UUUAUGACGCUCAUCCGCUGA 417 AGCGGAUGAGCGUCAUAAATT 867 IGF2R 7185UAUUUGUAGGACACGUUGGAA 418 CCAACGUGUCCUACAAAUATT 868 IGF2R 4447UACCCUGCCGAGGUUCACGGG 419 CGUGAACCUCGGCAGGGUATT 869 IGF2R 3706UAUCUGAGCACACUCAAACGT 420 GUUUGAGUGUGCUCAGAUATT 870 IGF2R 6422UCUUUGUACAGGUCAAUUCTA 421 GAAUUGACCUGUACAAAGATT 871 IGF2R 1306UUUGACUUGAGAGGUAUCGCT 422 CGAUACCUCUCAAGUCAAATT 872 IGF2R 6129UUGUGUUUCUGGACGAAUUTG 423 AAUUCGUCCAGAAACACAATT 873 IGF2R 5105UAGAGCUUCCAUUCCUCACGG 424 GUGAGGAAUGGAAGCUCUATT 874 IGF2R 4572UUCACUUGGCUCUCGCUGCAG 425 GCAGCGAGAGCCAAGUGAATT 875 IGF2R 5308UACCCGGCCGAUAUCUAUGGG 426 CAUAGAUAUCGGCCGGGUATT 876 IGF2R 3153UUCUCAAUUCCGACUGGCCTT 427 GGCCAGUCGGAAUUGAGAATT 877 IGF2R 9029UAUUACAGUAAAGUUGAUUGA 428 AAUCAACUUUACUGUAAUATT 878 IGF2R 1530UUAACACAGGCGUAUUCCGTG 429 CGGAAUACGCCUGUGUUAATT 879 IGF2R 8364AAAUGUGCUCUGUACGCCCAG 430 GGGCGUACAGAGCACAUUUTT 880 IGF2R 5400UAGUUGAAAUGCUUGUCCGCT 431 CGGACAAGCAUUUCAACUATT 881 IGF2R 6702UUGGCUCCAGAGCACGCCGGG 432 CGGCGUGCUCUGGAGCCAATT 882 IGF2R 8479UUCUCUGACACCUCAACUCCA 433 GAGUUGAGGUGUCAGAGAATT 883 IGF2R 4723UAAGGAGCUCAGAUCAAACAG 434 GUUUGAUCUGAGCUCCUUATT 884 IGF2R 4237UGAACAUUCAGUCAGAUCGAA 435 CGAUCUGACUGAAUGUUCATT 885 IGF2R 6203UAUAGUACGAGACUCCGUUGT 436 AACGGAGUCUCGUACUAUATT 886 IGF2R 753AUGAAUAGAGAAGUGUCCGGA 437 CGGACACUUCUCUAUUCAUTT 887 IGF2R 8554AUAAGCACAGUAAAGGUGGTA 438 CCACCUUUACUGUGCUUAUTT 888 IGF2R 5462UUAACAGCUUAGGCGUUCCCA 439 GGAACGCCUAAGCUGUUAATT 889 IGF2R 1460UUCCUUUCCCAUCGUUACCTG 440 GGUAACGAUGGGAAAGGAATT 890 IGF2R 5206AUUGAUGUAGAAAUCAGGGTT 441 CCCUGAUUUCUACAUCAAUTT 891 IGF2R 2559AUGUAGUAUUUCCUCCACGTG 442 CGUGGAGGAAAUACUACAUTT 892 IGF2R 8605UUAAGGCUUCUUAUAGGUCGA 443 GACCUAUAAGAAGCCUUAATT 893 IGF2R 4345AUUGAUGAGGUAGUGCUCCGG 444 GGAGCACUACCUCAUCAAUTT 894 IGF2R 1187AAAUAUAGGAUGAACCUCCGC 445 GGAGGUUCAUCCUAUAUUUTT 895 IGF2R 1184UAUAGGAUGAACCUCCGCUCT 446 AGCGGAGGUUCAUCCUAUATT 896 IGF2R 7190UUGAGUAUUUGUAGGACACGT 447 GUGUCCUACAAAUACUCAATT 897 IGF2R 7182UUGUAGGACACGUUGGAACTT 448 GUUCCAACGUGUCCUACAATT 898 IGF2R 2941AUCCCUUAUAGAGCAAGCCTG 449 GGCUUGCUCUAUAAGGGAUTT 899 IGF2R 3693UCAAACGUGAUCCUGGUGGAG 450 CCACCAGGAUCACGUUUGATT 900Chemical Modification of RNA Strand Nucleotides

The siRNA according to the invention may comprise at least one modifiednucleotide in at least one of the RNA strands. A range of potentialmodified nucleotides are disclosed elsewhere herein. Usefulmodifications and combinations of modifications for use according to theinvention are shown in Table 2:

TABLE 2  Chemical Modifications and Sequence Architecture Modification #Name Format 1 PS DNA o/h     NNNNNNNNNNNNNNNNNNNsnsnnsnsNNNNNNNNNNNNNNNNNNN 2 Full PS    NsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsN 3 RNA o/h  NNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNN 4 Blunt-endedNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN 5 2′-OMe o/h   NNNNNNNNNNNNNNNNNNNN^(p)N^(p) N^(p)N^(p) NNNNNNNNNNNNNNNNNNN 62′-OMe/2′F    NNNNNNNNNNNNNNNNNNNN^(p)N^(p)N^(p)N^(p)NNNNNNNNNNNNNNNNNNN 7 LNA (3-7     NNNNNNNNNNNNNNNNNNNsnsnincorporations nsnsNNNNNNNNNNNNNNNNNNN in ds region) N = any unmodifiedRNA nucleotide n = unmodified DNA nucleotide N^(p) = modified RNAnucleotide s = identifies phosphorthioate internucleoside linkage o/h =overhang

The following modifications added to the 3′ position of the 3′-terminusof the siRNA strands, sometimes referred to as a ‘3’ end cap′ are alsorecognized as useful embodiments of the invention and may be used withany of the siRNA according to the invention:

Specific compounds with activity according to the invention include thefollowing, shown in Table 3:

TABLE 3  Sequences and Chemistries of siRNA used in ExamplesSequence (N: RNA; dN: DNA; n:  Name strand2′-moe RNA; s: phosphorothioate) SEQ ID NO pGI3-siRNA guide strandUCG AAG UAC UCA GCG UAA 901 GdTdT complement CUU ACG CUG AGU ACU UCG 902strand AdTdT pGL3 MOE o/h guide strand CUU ACG CUG AGU ACU UCG Atst 903siRNA complement UCG AAG UAC UCA GCG UAA Gtst 904 strand pGI3-C3-siRNAguide strand UCG AAG UAC UCA GCG UAA G-C3 905 complementCUU ACG CUG AGU ACU UCG A-C3 906 strand pGI3-C3-MOE- guide strandUCG AAG UAC UCA GCG UAa g-C3 907 siRNA complementCUU ACG CUG AGU ACU UCg a-C3 908 strand VEGFR2-siRNA1 guide strandUUG AGG UUU GAA AUC GAC 909 CdCdT complement GGU CGA UUU CAA ACC UCA 910strand AdTdT VEGFR2-siRNA2 guide strand UAA UUU GUU CCU GUC UUC 911CdAdG complement GGA AGA CAG GAA CAA AUU 912 strand AdTdT siRNA controlguide strand ACG UGA CAC GUU CGG AGA 913 AdTdT complementUUC UCC GAA CGU GUC ACG 914 strand UdTdT VEGFR2-C3- guide strandUUG AGG UUU GAA AUC GAC C-C3 915 siRNA1 complementGGU CGA UUU CAA ACC UCA A-C3 916 strand VEGFR2-C3- guide strandUAA UUU GUU CCU GUC UUC C-C3 917 siRNA2 complementGGA AGA CAG GAA CAA AUU A-C3 918 strand C3-siRNA  guide strandACG UGA CAC GUU CGG AGA A-C3 919 control complementUUC UCC GAA CGU GUC ACG U-C3 920 strand VEGFR2-C3- guide strandUUG AGG UUU GAA AUC GAc c-C3 921 MOE-siRNA1 complementGGU CGA UUU CAA ACC UCa a-C3 922 strand VEGFR2-C3- guide strandUAA UUU GUU CCU GUC UUc c-C3 923 MOE-siRNA2 complementGGA AGA CAG GAA CAA AUu a-C3 924 strand Tie2-C3-MOE- guide strandUUC UUC UUU AAU UAA CAc c-C3 925 siRNA1 complementGGU GUU AAU UAA AGA AGa a-C3 926 strand Tie2-C3-MOE- guide strandUCU GAG UUU GUA AAU AUc g-C3 927 siRNA2 complementCGA UAU UUA CAA ACU CAg a-C3 928 strand C3-MOE-siRNA guide strandACG UGA CAC GUU CGG AGa a-C3 929 control complementUUC UCC GAA CGU GUC ACg t-C3 930 strand

EXAMPLES

The following Examples illustrate aspects of the invention, and are notintended to limit the embodiments included in the claims recited below.The results and discussion section further below refers to experimentsconducted according to the following protocols and employing thefollowing materials. Materials and protocols that are not specificallydescribed are considered to be routinely available to those skilled inthe art.

Example 1

Preparation of siRNAs.

Single strand siRNA derivatives were synthesized by standard 2′-O-TOMphosphoamidite technology and purified by Oasis® HLB Extraction Plates(Waters). Sense- and antisense stranded siRNA were mixed inhybridization buffer (100 mM potassium acetate, 2 mM magnesium acetate,30 mM Hepes, pH 7.6) heat-denatured at 90° C. for 3 min and annealed at37° C. for 60 min. 100 μM stock solutions of siRNA duplexes were storedat −20° C.

Example 2

Incubation in Serum and Analysis by IE-HPLC (LC-MS).

In a standard serum assay, 6 μL 20 μM of each siRNA were mixed with 54μL serum or CSF and heated at 37° C. in an incubator. 50 μL of thecooled mixture was loaded on an analytical DNA-pac PA-100 Column(Dionex) and analyzed with a NaCl gradient (0-0.6 M in 30 min) in a 1:10Acetonitrile:Buffer (20 mM sodium acetate, 1 mM magnesium acetate, pH6.5) solution.

For LC-MS analysis 100 μL (20 μM or 50 μM) each siRNA was mixed with 900μL sterile fetal bovine serum (GIBCO) incubated at 37° C. and separatedby HPLC as indicated previously (except of the NaCl gradient: 0M-0.36Min 9′/0.36M-0.6M in 12′). Degradation products were desalted on NAPcolumns and analyzed by LC-ESI⁻-MS.

Example 3

Incubation in Gastric Acid

To prepare a standard gastric acid assay, FVB and C57BL6 mice, weighing18 to 20 g (6 to 8 weeks old), were obtained from Charles RiverLaboratories (Les Oncins, France). Animals were sacrificed using CO₂,and then stomachs were quickly recovered. Gastric fluid as well asstomach contents were collected and pooled, then loaded on centrifugalfilter devices (Ultrafree MC, Millipores). Filter units were spun for 10minutes according to manufacturer's recommendations. The filtrate,corresponding to mouse gastric fluid, was recovered, aliquoted andfrozen prior further experiments.

For each assay, 20 μM of siRNA solutions were diluted in 9× volume ofgastric acid as above described and incubated at 37° C. for 0, 5, 10,15, 30, 60 and 120 min.

Example 4

Incubation in Intestinal Lavage

To prepare a standard intestinal lavage assay, Male Wistar rat werefasted, anesthetized with isoflurane. Intestinal lavage was obtained byin situ perfusion of the small intestine (duodenum, jejunum, ileum) with10 mL saline (0.5 mL/min) followed by 20 mL water (1 mL/min) Outletcollected was centrifuged (3000×g, 15 min, 22° C.), and supernatantpassed through a 1.2-nm filter and stored at −20° C.

For each assay, 20 μM siRNA solutions were diluted in 9× volume ofintestinal lavage and incubated at 37° C. for 0, 15, 30, 60, 180 and 360min.

Example 5

Incubation in Mouse Liver Microsomes

In a standard liver microsome assay, to 10 μl of a 250 μM solution ofsiRNA were added 25 μl of mouse liver microsomes (GEntest 452701 Charge11) at 20 mg protein/ml, 365 μl of 100 mM phosphate buffer (pH 7.4), 50μl of UDPGA cofactor (24 mM in water), 50 μl of NADPH. Incubation wasquenched by freezing at t=0 min and t=60 min.

Example 6

Incubation in Rat S12 Supernatant

For a standard rat S12 supernatant assay, 10 μl of a 250 μM solution ofsiRNA were added to 17 μl of rat liver S12 at 29.9 mg protein/ml, 373 μlof 100 mM phosphate buffer (pH 7.4), 50 μl of UDPGA cofactor (24 mM inwater), 50 μl of NADPH. Incubation was quenched by freezing at t=0 minand t=60 min.

Example 7

Incubation in Mouse Serum

For a standard incubation in mouse serum, 20 μM siRNA solutions werediluted in 9× volume of murine serum (Harlan nude mouse) and incubatedat 37° C. for 0, 15, 30, 60, 180 and 360 min.

Example 8

Gel Electrophoresis Stability Assay

A 10 μL aliquot of incubation solution was taken immediately aftershaking and shock-frozen on dry ice, the mixtures were incubated at 37°C. and aliquots were shock frozen at various time points. Aliquots werethawed in 30 μL (15 μL respectively) Loading Buffer (Elchrom Sc., Cham,Switzerland) and separated on a SF50 gels (Elchrom Sc., Cham,Switzerland) at 120 V, 8° C. for 240 min. Bands were stained with SYBRGold (Molecular Probes) and picture were taken with a BIORAD ChemiDoc™XRS system.

Example 9

Cell Culture

The mouse immortalized endothelial cell line MS1 (ATCC CRL-2279) wasgrown in DMEM high glucose (4.5 g/1) supplemented with L-Glutamine and10% heat-inactivated FCS (AMIMED, Switzerland) on 1.5% Gelatine-coatedculture dishes. MS1 cells were transfected in 24 well-format with siRNAusing HiPerfect (QIAGEN) according to manufacturer procedure(tetraplicate, final siRNA concentration was 10 nM or as indicated).

Example 10

FACS Analysis

Non-transfected and siRNA transfected MS1 cells were analyzed by FACSfor VEGFR2 levels. Briefly, cells were trypsinized from duplicate ortriplicate wells, pooled for each conditions, then washed twice withPBS+10% FCS and incubated 10 minutes on ice prior addition ofRPE-conjugated anti-VEGFR2 Ab (1 μg/10⁶ cells; Avas 12α1, BDPharmingen). RPE-labeled isotype IgG2α were used as FACS control (BDPharmingen). FACS acquisition and analysis were performed on aFACScalibur using Cell Quest Software (Becton-Dickinson).

Example 11

Animal Studies

Female FVB mice (6 to 8 weeks old), were obtained from Charles RiverLaboratories (Les Oncins, France). Mice were identified via ear markingsand kept in groups (6 animals per cage) under normal conditions andobserved daily. Six mice were used per treatment group and all animalexperiments were performed in strict adherence to the Swiss law foranimal protection.

The reference chamber model has been described in publications (e.g.Wood J, Bold G, Buchdunger E, et al. PTK787/ZK 222584, a novel andpotent inhibitor of vascular endothelial growth factor receptor tyrosinekinases, impairs vascular endothelial growth factor-induced responsesand tumor growth after oral administration. Cancer Res 2000; 60:2178-89)In brief, porous tissue chambers made of perfluoro-alkoxy-Teflon(Teflon®-PFA, 21 mm×8 mm diameter, 550 μl volume) were filled with 0.8%agar (BBL® Nr. 11849, Becton Dickinson, Meylan, France) and 20 U/mlheparin, (Novo Nordisk A/S, Bagsvaerd, Denmark) supplemented with orwithout 3 μg/ml recombinant human VEGF and siRNAs as indicated.Solutions were maintained at 42° C. prior the filling procedure. Micewere anesthetized using 3% Isoflurane (Forene®, Abbott AG, Cham,Switzerland) inhalation. For subcutaneous implantation, a small skinincision was made at the base of the tail to allow the insertion of animplant trocar. The chamber was implanted under aseptic conditionsthrough the small incision onto the back of the animal. The skinincision was closed by wound clips (Autoclip 9 mm Clay Adams). Dependingon the required dose, siRNAs were diluted in “injectable quality grade”0.9% saline solution then delivered to animals either i.p. (200 μL/dose)or p.o. by gavage (100 μL/dose). The mice were receiving the first dose2 to 4 hours before implanting chambers; then treated daily for 2 days.If not otherwise indicated, mice were sacrificed three days afterimplantation, chambers excised and the vascularized fibrous tissueformed around each implant carefully removed. Body weight was used tomonitor the general condition of the mice. Statistical analysis was doneusing one-way ANOVA followed by Dunnett test.

Example 12

B16 Melanoma Xenograft Model

The syngeneic B16/BL6 murine melanoma model, previously identified to beresponsive to antiangiogenic therapy (e.g. LaMontagne K,Littlewood-Evans A, Schnell C, O'Reilly T, Wyder L, Sanchez T, Probst B,Butler J, Wood A, Liau G, Billy E, Theuer A, Hla T, Wood J. Antagonismof sphingosine-1-phosphate receptors by FTY720 inhibits angiogenesis andtumor vascularization. Cancer Res. 2006 Jan. 1; 66(1):221-31), was usedto evaluate the antitumor activity of standard or modified siRNAs. Tumorcells (1 μL, 5×10⁴/μL) were injected intradermally into the dorsal pinnaof both ears of syngeneic female C57BL/6 mice. Measurements of primarytumor area (mm²) were carried out on days 7, 14, and 21 after tumor cellinoculation using computer-assisted image analysis software (KS-400 3.0imaging system, Zeiss) and a specifically designed macro. From days 7 to21, mice were receiving siRNAs diluted in “injectable quality grade”0.9% saline solution either i.p. (200 μL/dose) or p.o. by gavage (100μL/dose) once a day. Mice were sacrificed on day 21, and cranial lymphnode metastases were weighed and then frozen.

In these results, actual siRNA sequences and chemistries employed may bedetermined by reference to Table 3.

Wild-type siRNAs are Degraded in Mouse Serum from Both 3′-ends

Oligonucleotide degradation by nucleases is predominantly3′-exonucleolytic. Modification of antisense oligonucleotides at theirtermini by the introduction of aromatic or lipophilic residues delaystheir nucleolytic degradation¹⁷. To verify whether this metabolicpathway would also be dominant for siRNA, we incubated at 37° C. aunmodified siRNA (wild-type siRNA) in mouse serum for up to 3 hours.

The unmodified siRNA sequence employed was pGl3-siRNA (see Table 3)

The mixtures were analyzed with Strong Anion Exchange HPLC at t=0 min.,t=30 min, t=180 min.

As shown in FIGS. 1a, 1b and 1c , at t=30 min, a well defined peakcorresponding to blunt ended siRNA was observed. By t=3 h substantialdegradation is observed. FIG. 1d and 1 e illustrate the metabolitesidentified by HPLC-ESI-MS analysis. This analysis revealed the presenceof several metabolites corresponding to the loss of the 3′ overhangs andof the 3′-terminal first base pairing ribonucleotide on both strands.Digestion of the 5′-terminal ribonucleotide of the guide strand was alsoobserved.

FIG. 1 suggests the degradation pathway of unmodified siRNAs in serum.DNA overhangs are first digested, possibly by 3′-exonucleases. In theLC-MS, additional metabolites were also detected which correspond to theloss of the first base-pairing 3′-ribonucleotide of both strands andalso the first 5′-base-pairing ribonucleotide of the guide strand.

3′-modified siRNAs are Stable Through the GI Tract

siRNAs with 2′-methoxyethyl ribonucleotides overhangs (MOE o/h siRNA),blunt-ended siRNAs 3′-capped with a hydroxypropoxy phosphodiester moiety(C3-siRNA), and hydroxypropoxy phosphodiester 3′-capped siRNAs where thetwo first base paring nucleotide at 3′-end of each strand were modifiedby 2′-methoxyethyl ribonucleotides residues (C3-MOE siRNA) weresynthesized. These compounds are illustrated schematically in FIG. 2.

First siRNAs were incubated in mouse gastric acid for 2 h (FIG. 3). Nodegradation was observed in the cases of C3 siRNA and C3-MOE sRNA, whiledegradation of wild-type siRNA was observed after 30 minutes.

Stability in intestinal fluid obtained from intestinal lavage of ratsrevealed almost complete degradation of wild-type siRNA after 15 minuteswhereas parent compound in the MOE o/h siRNA, C3-siRNA and C3-Moe siRNAwere observed for 60 minutes. (FIG. 4)

Stability in liver was evaluated using a liver microsome assay and a S12assay (representative of liver cytosolic enzymatic activity). Resultsare shown in FIG. 5. In both cases, no degradation was observed after 60minutes of incubation.

Finally, siRNAs were tested in mouse serum by incubation at 2 micromolarfor up to 6 hours at 37° C. (results in FIG. 6). Parent compoundstability was followed by gel electrophoresis. In the cases of modifiedsiRNAs (C3 siRNA, C3-MOE siRNA of MOE o/h siRNA), no significantdegradation was observed while the wild-type siRNA

This study indicate that wild type (unmodified) siRNAs are metabolizedin mouse gastric acid and in mouse serum. In case of 3′-ends modifiedsiRNAs, no degradation was observed in the GI tract. Therefore it islikely that 3′-modified siRNAs will have a higher oral bioavailabilitythan wild-type siRNAs

Systemically Delivered 3′-modified siRNAs are More Active in an in vivoGrowth Factor Induced Angiogenesis Model¹⁸.

Firstly, the ability of modified siRNAs (C3-siRNA and CE-MOE siRNA) todown regulate a target gene was checked in cellulo by measuring VEGFR2surface level of MS1 cells transfected with anti-VEGFR2 siRNAs.

Pools of 2 anti VEGFR2 siRNAs as wild-type siRNAs, C3-siRNAs and C3-MOEsiRNAs were administered intraperitoneally. Results are shown in FIG. 7.Pooled Wild type siRNAs reduced significantly the VEGF inducedvascularization at the higher dose of 25 micrograms per mice per day.The same level of inhibition was observed at a 5-fold lower dose withC3-siRNA. In the case of the C3-MOE siRNAs pool, significant reductionof vascularized tissue weight was observed at all tested doses includingthe lowest 0.2 microgram per mouse per day.

FIGS. 8a and 8b show that, when given intraperitoneally, both VEGFR2-C3and C3-MOE siRNAs were active at below 1 microgram per mouse per daydose.

In vivo testing of anti-VEGFR2 C3-MOE siRNA given intraperitoneally(i.p.) in a B16 homograft melanoma tumor mouse model. FIG. 9a shows thati.p. treatment with modified VEGFR2-C3-MOE-siRNA significantly reducestumour development. FIG. 9b also shows that i.p. injection ofVEGFR2-C3-MOE-siRNA at 20 ug per mouse results in significant inhibitionof tumour growth.

Oral Delivery of siRNA for Treatment of Angiogenic Disorders

FIG. 10 shows that given orally, at a dose of 20 micrograms per mouseper day, the VEGFR2-C3-MOE-siRNA1 reduced vascularization weight down tobasal level (e.g. weight without growth factor induction). Actual siRNAsequences used are referred to in Table 3.

Anti Tie2 C3-MOE siRNAs were also tested in the growth factor inducedangiogenesis model under both intraperitoneal and oral deliveries. FIGS.11a and 11b show that given orally, both C3-MOE siRNAs directed at Tie2were active at 20 microgram per mouse per day. Actual siRNA sequencesused may be determined by reference to Table 3.

The data shows that 3′-end modified siRNAs with or without additionalinternal modifications are able to demonstrate therapeutic effect atreasonable doses upon oral administration.

REFERENCES

-   -   1. a) Y. Tomari et al. Genes and Development 19 (2005),        517; b) P. Shankar et al. JAMA 11 (2005), 1367; c) Y. Dorsett et        al. Nature Reviews 3 (2004), 318    -   2. a) P. D. Zamore et al. Cell 101, (2000), 25; b) S. M. Hammond        et al. Nature 404 (2000), 293    -   3. a) G. Meister et al. Molecular Cell 15 (2004), 185.    -   4. S. M. Elbashir et al. Genes Dev. 15 (2001), 188.    -   5. S. J. Reich et al. Molecular Vision 9 (2003), 210.    -   6. a) Dorn et al. Nucleic Acids Research 32 (2004),        e49; b) D. R. Thakker et al. PNAS 101 (2004), 17270; c) D. R.        Thakker et al. Molecular Psychiatry 10 (2005), 714    -   7. V. Bitko et al. Nature Medicine 11 (2005), 50.    -   8. E. Song et al. Nature Medicine 9 (2003), 347.    -   9. D. A. Braasch et al. Biochemistry 42 (2003), 7967.    -   10. Harborth, Antisense Nucleic Acid Drug Devt, 2003    -   11. A. H. S. Hall et al. Nucleic Acids Research 32 (2004), 5991.    -   12. M. Amarzguioui et al. Nucleic Acids Research 31 (2003), 589.    -   13. F. Czauderna et al. Nucleic Acids Research 31 (2003), 2705.    -   14. T. Prakash et al. Journal of Medicinal Chemistry 48 (2005),        4247.    -   15. J. Elmen et al. Nucleic Acids Research 33 (2005), 439.    -   16. A. S. Boutorin, L. V. Guskova, E. M. Ivanova, N. D.        Kobetz, V. F. Zafytova, A. S. Ryte, L. V. Yurchenko and V. V.        Vlassov FEBS Lett. 254 (1989), p. 129    -   17. J. Wood et al. Cancer Research 60 (2000), 2178.    -   18. K. LaMontagne et al. Cancer Res. 66 (2006), 221.

We claim:
 1. Short interfering ribonucleic acid (siRNA), comprising twostrands that are complementary to each other over at least 15nucleotides, wherein each strand comprises 49 nucleotides or less,wherein the first two base-pairing nucleotides at the 3′ end of eachstrand are 2′-methoxyethyl ribonucleotide residues, and wherein a3′-terminus of at least one strand containing a 2′-methoxyethylribonucleotide comprises a modification at the 3′ carbon, wherein themodification is selected from the group consisting of:


2. The siRNA according to claim 1, wherein at least one additionalnucleotide is modified with a modification.
 3. The siRNA of claim 2,wherein the modification is selected from 2′-OMe, 2′-F, 2′-Oallyl,2′-MOE, Locked Nucleic Acid, phosphorothioate, and boranophosphate. 4.The siRNA of claim 1, wherein each strand is 19 nucleotides.
 5. ThesiRNA according to claim 1, wherein said siRNA comprises modifiednucleotides selected from among nucleotides having a modifiedinternucleoside linkage selected from among phosphorothioate,phosphorodithioate, phosphoramidate, boranophosphonoate, and amidelinkages.
 6. The siRNA according to claim 1, wherein said two RNAstrands are fully complementary to each other.
 7. The siRNA according toclaim 1, comprising a 1 to 9 nucleotide overhang on at least one of the5′ end or 3′ end.
 8. The siRNA according to claim 1, wherein the twostrands are complementary to each other for 19 nucleotides.
 9. The siRNAaccording to claim 1, wherein one end of the siRNA is blunt-ended. 10.The siRNA according to claim 1, wherein both ends of the siRNA areblunt-ended.
 11. The siRNA according to claim 1, wherein both ends areblunt-ended, and the two strands are complementary to each other for 19nucleotides.
 12. A composition comprising a siRNA of claim 1 and apharmaceutically acceptable carrier.
 13. Short interfering ribonucleicacid (siRNA) comprising two strands that are complementary to each otherover at least 15 nucleotides, wherein each strand comprises 49nucleotides or less, and wherein the first two base-pairing nucleotidesat the 3′ end of each strand are 2′-methoxyethyl (2′-MOE) ribonucleotideresidues, and wherein at least one additional nucleotide is modifiedwith a modification selected from 2′-OMe, 2′-F, 2′-O-allyl, 2′-MOE,Locked Nucleic Acid, phosphorothioate, and boranophosphate, and whereina 3′-terminus of at least one strand containing a 2′-methoxyethylribonucleotide comprises a modification at the 3′ carbon, wherein themodification is selected from the group consisting of triethyleneglycol, cyclohexyl, phenyl, and adamantane.